Compositions and Methods for Inhibiting Expression of Eg5 and VEGF Genes

ABSTRACT

This invention relates to compositions containing double-stranded ribonucleic acid (dsRNA) in a SNALP formulation, and methods of using the compositions to inhibit the expression of the Eg5 and Vascular Endothelial Growth Factor (VEGF), and methods of using the compositions to treat pathological processes mediated by Eg5 and VEGF expression, such as cancer.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 13/442,809, filed Apr. 9, 2012 (allowed), which is a continuation of U.S. patent application Ser. No. 12/552,207, filed Sep. 1, 2009 (abandoned), which is a continuation of PCT Application No. PCT/US2009/036233, filed Mar. 5, 2009 which claims the benefit of U.S. Provisional Application No. 61/034,019, filed Mar. 5, 2008, and U.S. Provisional Application No. 61/083,367, filed Jul. 24, 2008, and U.S. Provisional Application No. 61/086,381, filed Aug. 5, 2008, and U.S. Provisional Application No. 61/112,079, filed Nov. 6, 2008, and U.S. Provisional Application No. 61/150,664, filed Feb. 6, 2009 which are hereby incorporated in their entirety by reference.

FIELD OF THE INVENTION

This invention relates to compositions containing double-stranded ribonucleic acid (dsRNA), and their use in mediating RNA interference to inhibit the expression of a combination of genes, e.g., the Eg5 and Vascular Endothelial Growth Factor (VEGF) genes formulated in SNALP, and the use of the compositions to treat pathological processes mediated by Eg5 and VEGF expression, such as cancer.

REFERENCE TO SEQUENCE LISTING

This application includes a Sequence Listing submitted electronically as a text file named 28725_sequence_listing.txt, created on Feb. 2, 2015, with a size of 640,482 bytes. The sequence listing is incorporated by reference.

BACKGROUND OF THE INVENTION

The maintenance of cell populations within an organism is governed by the cellular processes of cell division and programmed cell death. Within normal cells, the cellular events associated with the initiation and completion of each process is highly regulated. In proliferative disease such as cancer, one or both of these processes may be perturbed. For example, a cancer cell may have lost its regulation (checkpoint control) of the cell division cycle through either the overexpression of a positive regulator or the loss of a negative regulator, perhaps by mutation.

Alternatively, a cancer cell may have lost the ability to undergo programmed cell death through the overexpression of a negative regulator. Hence, there is a need to develop new chemotherapeutic drugs that will restore the processes of checkpoint control and programmed cell death to cancerous cells.

One approach to the treatment of human cancers is to target a protein that is essential for cell cycle progression. In order for the cell cycle to proceed from one phase to the next, certain prerequisite events must be completed. There are checkpoints within the cell cycle that enforce the proper order of events and phases. One such checkpoint is the spindle checkpoint that occurs during the metaphase stage of mitosis. Small molecules that target proteins with essential functions in mitosis may initiate the spindle checkpoint to arrest cells in mitosis. Of the small molecules that arrest cells in mitosis, those which display anti-tumor activity in the clinic also induce apoptosis, the morphological changes associated with programmed cell death. An effective chemotherapeutic for the treatment of cancer may thus be one which induces checkpoint control and programmed cell death. Unfortunately, there are few compounds available for controlling these processes within the cell. Most compounds known to cause mitotic arrest and apoptosis act as tubulin binding agents. These compounds alter the dynamic instability of microtubules and indirectly alter the function/structure of the mitotic spindle thereby causing mitotic arrest. Because most of these compounds specifically target the tubulin protein which is a component of all microtubules, they may also affect one or more of the numerous normal cellular processes in which microtubules have a role. Hence, there is also a need for agents that more specifically target proteins associated with proliferating cells.

Eg5 is one of several kinesin-like motor proteins that are localized to the mitotic spindle and known to be required for formation and/or function of the bipolar mitotic spindle. Recently, there was a report of a small molecule that disturbs bipolarity of the mitotic spindle (Mayer, T. U. et. al. 1999. Science 286(5441) 971-4, herein incorporated by reference). More specifically, the small molecule induced the formation of an aberrant mitotic spindle wherein a monoastral array of microtubules emanated from a central pair of centrosomes, with chromosomes attached to the distal ends of the microtubules. The small molecule was dubbed “monastrol” after the monoastral array. This monoastral array phenotype had been previously observed in mitotic cells that were immunodepleted of the Eg5 motor protein. This distinctive monoastral array phenotype facilitated identification of monastrol as a potential inhibitor of Eg5. Indeed, monastrol was further shown to inhibit the Eg5 motor-driven motility of microtubules in an in vitro assay. The Eg5 inhibitor monastrol had no apparent effect upon the related kinesin motor or upon the motor(s) responsible for golgi apparatus movement within the cell. Cells that display the monoastral array phenotype either through immunodepletion of Eg5 or monastrol inhibition of Eg5 arrest in M-phase of the cell cycle. However, the mitotic arrest induced by either immunodepletion or inhibition of Eg5 is transient (Kapoor, T. M., 2000. J Cell Biol 150(5) 975-80). Both the monoastral array phenotype and the cell cycle arrest in mitosis induced by monastrol are reversible. Cells recover to form a normal bipolar mitotic spindle, to complete mitosis and to proceed through the cell cycle and normal cell proliferation. These data suggest that an inhibitor of Eg5 which induced a transient mitotic arrest may not be effective for the treatment of cancer cell proliferation. Nonetheless, the discovery that monastrol causes mitotic arrest is intriguing and hence there is a need to further study and identify compounds which can be used to modulate the Eg5 motor protein in a manner that would be effective in the treatment of human cancers. There is also a need to explore the use of these compounds in combination with other antineoplastic agents.

VEGF (also known as vascular permeability factor, VPF) is a multifunctional cytokine that stimulates angiogenesis, epithelial cell proliferation, and endothelial cell survival. VEGF can be produced by a wide variety of tissues, and its overexpression or aberrant expression can result in a variety disorders, including cancers and retinal disorders such as age-related macular degeneration and other angiogenic disorders.

Recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene.

SUMMARY OF THE INVENTION

Disclosed are compositions having two double-stranded ribonucleic acids (dsRNA) for inhibiting the expression of a human kinesin family member 11 (Eg5/KSP) and a human VEGF gene in a cell. The dsRNAs are formulated in a stable nucleic acid lipid particle (SNALP). Also disclosed are method for using the composition to decrease expression of Eg5/KSP and/or VEGF in a cell, and method of treatment of a disease, e.g., liver cancer, using the compositions of the invention.

Accordingly, disclosed herein is a composition having a first double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human kinesin family member 11 (Eg5/KSP) gene in a cell and a second dsRNA for inhibiting expression of a human VEGF in a cell, wherein both said first and said second dsRNA are formulated in a stable nucleic acid lipid particle (SNALP); said first dsRNA consists of a first sense strand and a first antisense strand, and said first sense strand has a first sequence and said first antisense strand has a second sequence complementary to at least 15 contiguous nucleotides of SEQ ID NO:1311 (5′-UCGAGAAUCUAAACUAACU-3′), wherein said first sequence is complementary to said second sequence and wherein said first dsRNA is between 15 and 30 base pairs in length; and said second dsRNA consists of a second sense strand and a second antisense strand, said second sense strand having a third sequence and said second antisense strand having a fourth sequence complementary to at least 15 contiguous nucleotides of SEQ ID NO:1538 (5′-GCACAUAGGAGAGAUGAGCUU-3′), wherein said third sequence is complementary to said fourth sequence and wherein each strand is between 15 and 30 base pairs in length.

In some embodiments, the first antisense strand has a second sequence complementary to SEQ ID NO:1311 (5′-UCGAGAAUCUAAACUAACU-3′) and the second antisense strand has a fourth sequence complementary to SEQ ID NO:1538 (5′-GCACAUAGGAGAGAUGAGCUU-3′). In other embodiments, the first dsRNA consists of a sense strand consisting of SEQ ID NO:1534 (5′-UCGAGAAUCUAAACUAACUTT-3′) and an antisense strand consisting of SEQ ID NO:1535 (5′-AGUUAGUUUAGAUUCUCGATT-3′) and the second dsRNA consists of a sense strand consisting of SEQ ID NO:1536 (5′-GCACAUAGGAGAGAUGAGCUU-3′), and an antisense strand consisting of SEQ ID NO:1537 (5′-AAGCUCAUCUCUCCUAUGUGCUG-3′). In further embodiments, each strand is modified as follows to include a 2′-O-methyl ribonucleotide as indicated by a lower case letter “c” or “u” and a phosphorothioate as indicated by a lower case letter “s”: the first dsRNA consists of a sense strand consisting of SEQ ID NO:1240 (5′-ucGAGAAucuAAAcuAAcuTsT-3′), and an antisense strand consisting of SEQ ID NO:1241 (5′-AGUuAGUUuAGAUUCUCGATsT); the second dsRNA consists of a sense strand consisting of SEQ ID NO:1242 (5′-GcAcAuAGGAGAGAuGAGCUsU-3′) and an antisense strand consisting of SEQ ID NO:1243 (5′-AAGCUcAUCUCUCCuAuGuGCusG-3′).

In some embodiments, the first dsRNA contains two overhangs and the second dsRNA contains an overhang at the 3′ of the antisense and a blunt end at the 5′ end of the antisense strand.

The first and second dsRNA can have at least one modified nucleotide. For example, each dsRNA can have at least one modified nucleotide chosen from the group of: a 2′-O-methyl modified nucleotide, a nucleotide having a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. The modified nucleotide can be chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base having nucleotide. In some embodiments, the first and second dsRNA each comprise at least one 2′-O-methyl modified ribonucleotide and at least one nucleotide having a 5′-phosphorothioate group.

Each strand of each dsRNA can be, e.g., 19-23 bases in length, or, alternatively 21-23 bases in length. In one embodiment, each strand of the first dsRNA is 21 bases in length and the sense strand of the second dsRNA is 21 bases in length and the antisense strand of the second dsRNA is 23 bases in length.

In some embodiments, the first and second dsRNA are present in an equimolar ratio.

As described herein, the dsRNAs are formulated as SNALPS. In some embodiments, the SNALP formulation includes DLinDMA, cholesterol, DPPC, and PEG2000-C-DMA. For example, the SNALP can have the components in the proportions listed in Table 17.

The composition of the invention can be used to reduce expression of Eg5 and/or VAGF. In some embodiments, the composition of the invention, upon contact with a cell expressing Eg5, inhibits expression of Eg5 by at least 40, 50, 60, 70, 80, or by at least 90%. In other embodiments, the composition of the invention, upon contact with a cell expressing VEGF, inhibits expression of VEGF by at least 40, 50, 60, 70, 80, or by at least 90%. Administration of the composition to a cell can expression of both Eg5 and VEGF in said cell. The composition of claims 1-17, wherein the composition is administered in a nM concentration.

Administration of the composition of the invention to a cell can result in, e.g., an increase in mono-aster formation in the cell. Administration of the composition to a mammal can result in at least one effect selected from the group consisting of prevention of tumor growth, reduction in tumor growth, or prolonged survival in said mammal. The effect can be measured using at least one assay selected from the group consisting of determination of body weight, determination of organ weight, visual inspection, mRNA analysis, serum AFP analysis and survival monitoring. Included are compositions with these effect when administered in a nM concentration.

In a further embodiment the composition of the invention includes Sorafenib.

Also included in the invention are methods of suing the compositions of the invention. In one embodiment is are methods for inhibiting the expression of Eg5/KSP and VEGF in a cell by administering any of the compositions of the invention to the cell. Other embodiments are methods for preventing tumor growth, reducing tumor growth, or prolonging survival in a mammal in need of treatment for cancer by administering the composition to said mammal. In some embodiments the mammal has liver cancer, e.g., the mammal is a human with liver cancer. The method can include a further step of administering Sorafenib.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing liver weights as percentage of body weight following administration of SNALP-siRNAs in a Hep3B mouse model.

FIG. 2A is a graph showing the effects of SNALP-siRNAs on body weight in a Hep3B mouse model.

FIG. 2B is a graph showing the effects of SNALP-siRNAs on body weight in a Hep3B mouse model.

FIG. 2C is a graph showing the effects of SNALP-siRNAs on body weight in a Hep3B mouse model.

FIG. 2D is a graph showing the effects of SNALP-siRNAs on body weight in a Hep3B mouse model.

FIG. 3 is a graph showing the effects of SNALP-siRNAs on body weight in a Hep3B mouse model.

FIG. 4 is a graph showing the body weight in untreated control animals.

FIG. 5 is a graph showing the effects of control luciferase-SNALP siRNAs on body weight in a Hep3B mouse model.

FIG. 6 is a graph showing the effects of VSP-SNALP siRNAs on body weight in a Hep3B mouse model.

FIG. 7A is a graph showing the effects of SNALP-siRNAs on human GAPDH levels normalized to mouse GAPDH levels in a Hep3B mouse model.

FIG. 7B is a graph showing the effects of SNALP-siRNAs on serum AFP levels as measured by serum ELISA in a Hep3B mouse model.

FIG. 8 is a graph showing the effects of SNALP-siRNAs on human GAPDH levels normalized to mouse GAPDH levels in a Hep3B mouse model.

FIG. 9 is a graph showing the effects of SNALP-siRNAs on human KSP levels normalized to human GAPDH levels in a Hep3B mouse model.

FIG. 10 is a graph showing the effects of SNALP-siRNAs on human VEGF levels normalized to human GAPDH levels in a Hep3B mouse model.

FIG. 11A is a graph showing the effects of SNALP-siRNAs on mouse VEGF levels normalized to human GAPDH levels in a Hep3B mouse model.

FIG. 11B is a set of graphs showing the effects of SNALP-siRNAs on human GAPDH levels and serum AFP levels in a Hep3B mouse model.

FIG. 12A is a graph showing the effects of SNALP-siRNAs on tumor KSP, VEGF and GAPDH levels in a Hep3B mouse model.

FIG. 12B is a graph showing the effects of SNALP-siRNAs on tumor KSP, VEGF and GAPDH levels in a Hep3B mouse model.

FIG. 12C is a graph showing the effects of SNALP-siRNAs on tumor KSP, VEGF and GAPDH levels in a Hep3B mouse model.

FIG. 13A is a graph showing the effects of SNALP-siRNAs on survival in mice with hepatic tumors; treatment was started at 18 days after tumor cell seeding.

FIG. 13B is a graph showing the effects of SNALP-siRNAs on survival in mice with hepatic tumors; treatment was started 26 days after tumor cell seeding.

FIG. 14 is a graph showing the effects of SNALP-siRNAs on serum alpha fetoprotein (AFP) levels.

FIG. 15A is an image of H&E stained sections in tumor bearing animals (three weeks after Hep3B cell implantation) administered 2 mg/kg SNALP-VSP. Twenty four hours later, tumor bearing liver lobes were processed for histological analysis. Arrows indicate mono asters.

FIG. 15B is an image of H&E stained sections in tumor bearing animals (three weeks after Hep3B cell implantation) administered 2 mg/kg SNALP-Luc. Twenty four hours later, tumor bearing liver lobes were processed for histological analysis. Arrows indicate mono asters.

FIG. 16 is a flow diagram illustrating the manufacturing process of ALN-VSPDS01.

FIG. 17 is a cryo-transmission electron microscope (cryo-TEM) image of ALN-VSP02.

FIG. 18 is a flow diagram illustrating the manufacturing process of ALN-VSP02.

FIG. 19 is a graph illustrating the effects on survival of administration SNALP formulated siRNA and Sorafenib.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions and methods for inhibiting the expression of the Eg5 gene and VEGF gene in a cell or mammal using the dsRNAs. The dsRNAs are preferably packaged in a stable nucleic acid particle (SNALP). The invention also provides compositions and methods for treating pathological conditions and diseases, such as liver cancer, in a mammal caused by the expression of the Eg5 gene and VEGF genes. The dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).

The following detailed description discloses how to make and use the compositions containing dsRNAs to inhibit the expression of the Eg5 gene and VEGF genes, respectively, as well as compositions and methods for treating diseases and disorders caused by the expression of these genes, such as cancer. The pharmaceutical compositions featured in the invention include a dsRNA having an antisense strand comprising a region of complementarity which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an RNA transcript of the Eg5 gene, together with a pharmaceutically acceptable carrier. The compositions featured in the invention also include a dsRNA having an antisense strand having a region of complementarity which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an RNA transcript of the VEGF gene.

Accordingly, certain aspects of the invention provide pharmaceutical compositions containing the Eg5 and VEGF dsRNAs and a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of the Eg5 gene and the VEGF gene respectively, and methods of using the pharmaceutical compositions to treat diseases caused by expression of the Eg5 and VEGF genes.

I. DEFINITIONS

For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. “T” and “dT” are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g., deoxyribothymine. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences comprising such replacement moieties are embodiments of the invention.

As used herein, “Eg5” refers to the human kinesin family member 11, which is also known as KIF11, Eg5, HKSP, KSP, KNSL1 or TRIPS. Eg5 sequence can be found as NCBI GeneID:3832, HGNC ID: HGNC:6388 and RefSeq ID number:NM_(—)004523. The terms “Eg5” and “KSP” and “Eg5/KSP are used interchangeably

As used herein, VEGF, also known as vascular permeability factor, is an angiogenic growth factor. VEGF is a homodimeric 45 kDa glycoprotein that exists in at least three different isoforms. VEGF isoforms are expressed in endothelial cells. The VEGF gene contains 8 exons that express a 189-amino acid protein isoform. A 165-amino acid isoform lacks the residues encoded by exon 6, whereas a 121-amino acid isoform lacks the residues encoded by exons 6 and 7. VEGF145 is an isoform predicted to contain 145 amino acids and to lack exon 7. VEGF can act on endothelial cells by binding to an endothelial tyrosine kinase receptor, such as Flt-1 (VEGFR-1) or KDR/flk-1 (VEGFR-2). VEGFR-2 is expressed in endothelial cells and is involved in endothelial cell differentiation and vasculogenesis. A third receptor, VEGFR-3, has been implicated in lymphogenesis.

The various isoforms have different biologic activities and clinical implications. For example, VEGF145 induces angiogenesis and like VEGF189 (but unlike VEGF165) VEGF145 binds efficiently to the extracellular matrix by a mechanism that is not dependent on extracellular matrix-associated heparin sulfates. VEGF displays activity as an endothelial cell mitogen and chemoattractant in vitro and induces vascular permeability and angiogenesis in vivo. VEGF is secreted by a wide variety of cancer cell types and promotes the growth of tumors by inducing the development of tumor-associated vasculature. Inhibition of VEGF function has been shown to limit both the growth of primary experimental tumors as well as the incidence of metastases in immunocompromised mice. Various dsRNAs directed to VEGF are described in co-pending U.S. Ser. Nos. 11/078,073 and 11/340,080, which are hereby incorporated by reference in their entirety.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the Eg5/KSP and/or VEGF gene, including mRNA that is a product of RNA processing of a primary transcription product.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes of the invention.

“Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but not limited to, G:U Wobble or Hoogstein base pairing.

The terms “complementary”, “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide which is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide which is substantially complementary to a contiguous portion of the mRNA of interest (e.g., encoding Eg5/KSP and/or VEGF) including a 5′ UTR, an open reading frame (ORF), or a 3′ UTR. For example, a polynucleotide is complementary to at least a part of a Eg5 mRNA if the sequence is substantially complementary to a non-interrupted portion of a mRNA encoding Eg5.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop”. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker”. The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs. In general, the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include at least one non-ribonucleotide, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this specification, “dsRNA” may include chemical modifications to ribonucleotides, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. Any such modifications, as used in an siRNA type molecule, are encompassed by “dsRNA” for the purposes of this specification and claims.

As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-end of the other strand, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule. In some embodiments the dsRNA can have a nucleotide overhang at one end of the duplex and a blunt end at the other end.

The term “antisense strand” refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches may be in the internal or terminal regions of the molecule. Generally the most tolerated mismatches are in the terminal regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA agent or a plasmid from which an iRNA agent is transcribed.

“Introducing into a cell”, when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be “introduced into a cell”, wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection.

The terms “silence” and “inhibit the expression of” “down-regulate the expression of,” “suppress the expression of” and the like in as far as they refer to the Eg5 and/or VEGF gene, herein refer to the at least partial suppression of the expression of the Eg5 gene, as manifested by a reduction of the amount of Eg5 mRNA and/or VEGF mRNA which may be isolated from a first cell or group of cells in which the Eg5 and/or VEGF gene is transcribed and which has or have been treated such that the expression of the Eg5 and/or VEGF gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}{\mspace{11mu} \;}{treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}{\mspace{11mu} \;}{control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to Eg5 and/or VEGF gene expression, e.g. the amount of protein encoded by the Eg5 and/or VEGF gene which is produced by a cell, or the number of cells displaying a certain phenotype, e.g. apoptosis. In principle, target gene silencing can be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given dsRNA inhibits the expression of the Eg5 gene by a certain degree and therefore is encompassed by the instant invention, the assay provided in the Examples below shall serve as such reference.

For example, in certain instances, expression of the Eg5 gene (or VEGF gene) is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of the double-stranded oligonucleotide of the invention. In some embodiments, the Eg5 and/or VEGF gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide of the invention. In other embodiments, the Eg5 and/or VEGF gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide of the invention. The Tables and Example below provides values for inhibition of expression using various Eg5 and/or VEGF dsRNA molecules at various concentrations.

As used herein in the context of Eg5 expression (or VEGF expression), the terms “treat”, “treatment”, and the like, refer to relief from or alleviation of pathological processes mediated by Eg5 and/or VEGF expression. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by Eg5 and/or VEGF expression), the terms “treat”, “treatment”, and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition, such as the slowing and progression of hepatic carcinoma.

As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes mediated by Eg5 and/or VEGF expression or an overt symptom of pathological processes mediated by Eg5 and/or VEGF expression. The specific amount that is therapeutically effective can be readily determined by ordinary medical practitioner, and may vary depending on factors known in the art, such as, e.g. the type of pathological processes mediated by Eg5 and/or VEGF expression, the patient's history and age, the stage of pathological processes mediated by Eg5 and/or VEGF expression, and the administration of other anti-pathological processes mediated by Eg5 and/or VEGF expression agents.

As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. As described in more detail below, such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.

II. DOUBLE-STRANDED RIBONUCLEIC ACID (DSRNA)

As described in more detail below, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the Eg5 and/or VEGF gene in a cell or mammal, wherein the dsRNA comprises an antisense strand comprising a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of the Eg5 and/or VEGF gene, and wherein the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and wherein said dsRNA, upon contact with a cell expressing said Eg5 and/or VEGF gene, inhibits the expression of said Eg5 and/or VEGF gene.

The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.

The dsRNA comprises two strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of the Eg5 and/or VEGF gene, the other strand (the sense strand) comprises a region which is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 base pairs in length. In other embodiments the duplex structure is 25-30 base pairs in length.

In one embodiment the duplex is 19 base pairs in length. In another embodiment the duplex is 21 base pairs in length. When two different siRNAs are used in combination, the duplex lengths can be identical or can differ. For example, a composition can include a first dsRNA targeted to Eg5 with a duplex length of 19 base pairs and a second dsRNA targeted to VEGF with a duplex length of 21 base pairs.

Similarly, the region of complementarity to the target sequence is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 nucleotides in length. In other embodiments the region of complementarity is 25-30 nucleotides in length.

In one embodiment the region of complementarity is 19 nucleotides in length. In another embodiment the region of complementarity is 21 nucleotides in length. When two different siRNAs are used in combination, the region of complementarity can be identical or can differ. For example, a composition can include a first dsRNA targeted to Eg5 with a region of complementarity of 19 nucleotides and a second dsRNA targeted to VEGF with a region of complementarity of 21 nucleotides.

Each strand of the dsRNA of invention is generally between 15 and 30, or between 18 and 25, or 18, 19, 20, 21, 22, 23, or 24 nucleotides in length. In other embodiments, each is strand is 25-30 base pairs in length. Each strand of the duplex can be the same length or of different lengths. When two different siRNAs are used in combination, the lengths of each strand of each siRNA can be identical or can differ. For example, a composition can include a dsRNA targeted to Eg5 with a sense strand of 21 nucleotides and an antisense strand of 21 nucleotides, and a second dsRNA targeted to VEGF with a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides.

The dsRNA of the invention can include one or more single-stranded overhang(s) of one or more nucleotides. In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. In another embodiment, the antisense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ end and the 5′ end over the sense strand. In further embodiments, the sense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ end and the 5′ end over the antisense strand.

A dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties than the blunt-ended counterpart. In some embodiments the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. A dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Generally, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA can also have a blunt end, generally located at the 5′-end of the antisense strand. Such dsRNAs can have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

As described in more detail herein, the composition of the invention includes a first dsRNA targeting Eg5 and a second dsRNA targeting VEGF. The first and second dsRNA can have the same overhang architecture, e.g., number of nucleotide overhangs on each strand, or each dsRNA can have a different architecture. In one embodiment, the first dsRNA targeting Eg5 includes a 2 nucleotide overhang at the 3′ end of each strand and the second dsRNA targeting VEGF includes a 2 nucleotide overhang on the 3′ end of the antisense strand and a blunt end at the 5′ end of the antisense strand (e.g., the 3′ end of the sense strand).

In one embodiment, the Eg5 gene targeted by the dsRNA of the invention is the human Eg5 gene. In one embodiment, the antisense strand of the dsRNA targeting Eg5 comprises at least 15 contiguous nucleotides of one of the antisense sequences of Table 1-3. In specific embodiments, the first sequence of the dsRNA is selected from one of the sense strands of Tables 1-3 and the second sequence is selected from the group consisting of the antisense sequences of Tables 1-3. Alternative antisense agents that target elsewhere in the target sequence provided in Tables 1-3 can readily be determined using the target sequence and the flanking Eg5 sequence. In some embodiments the dsRNA targeted to Eg5 will comprise at least two nucleotide sequence selected from the groups of sequences provided in Tables 1-3. One of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of the Eg5 gene. As such, the dsRNA will comprises two oligonucleotides, wherein one oligonucleotide is described as the sense strand in Tables 1-3 and the second oligonucleotide is described as the antisense strand in Tables 1-3

In embodiments using a second dsRNA targeting VEGF, such agents are exemplified in the Examples, Tables 4a and 4b, and in co-pending U.S. Ser. Nos. 11/078,073 and 11/340,080, herein incorporated by reference. In one embodiment the dsRNA targeting VEGF has an antisense strand complementary to at least 15 contiguous nucleotides of the VEGF target sequences described in Table 4a. In other embodiments, the dsRNA targeting VEGF comprises one of the antisense sequences of Table 4b, or one of the sense sequences of Table 4b, or comprises one of the duplexes (sense and antisense strands) of Table 4b.

The skilled person is well aware that dsRNAs comprising a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer dsRNAs can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 1-3, the dsRNAs of the invention can comprise at least one strand of a length of minimally 21 nt. It can be reasonably expected that shorter dsRNAs comprising one of the sequences of Tables 1-3 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs comprising a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 1-3, and differing in their ability to inhibit the expression of the Eg5 gene in a FACS assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further dsRNAs that cleave within the target sequence provided in Tables 1-3 can readily be made using the Eg5 sequence and the target sequence provided. Additional dsRNA targeting VEGF can be designed in a similar matter using the sequences disclosed in Tables 4a and 4b, the Examples and co-pending U.S. Ser. Nos. 11/078,073 and 11/340,080, herein incorporated by reference.

In addition, the RNAi agents provided in Tables 1-3 identify a site in the Eg5 mRNA that is susceptible to RNAi based cleavage. As such the present invention further includes RNAi agents, e.g., dsRNA, that target within the sequence targeted by one of the agents of the present invention. As used herein a second RNAi agent is said to target within the sequence of a first RNAi agent if the second RNAi agent cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first RNAi agent. Such a second agent will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Tables 1-3 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in the Eg5 gene. For example, the last 15 nucleotides of SEQ ID NO:1 combined with the next 6 nucleotides from the target Eg5 gene produces a single strand agent of 21 nucleotides that is based on one of the sequences provided in Tables 1-3. Additional RNAi agents, e.g., dsRNA, targeting VEGF can be designed in a similar matter using the sequences disclosed in Tables 4a and 4b, the Examples and co-pending U.S. Ser. Nos. 11/078,073 and 11/340,080, herein incorporated by reference.

The dsRNA of the invention can contain one or more mismatches to the target sequence. In a preferred embodiment, the dsRNA of the invention contains no more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand which is complementary to a region of the Eg5 gene, the dsRNA generally does not contain any mismatch within the central 13 nucleotides. The methods described within the invention can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of the Eg5 gene. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of the Eg5 gene is important, especially if the particular region of complementarity in the Eg5 gene is known to have polymorphic sequence variation within the population.

Modifications

In yet another embodiment, the dsRNA is chemically modified to enhance stability. The nucleic acids of the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Specific examples of preferred dsRNA compounds useful in this invention include dsRNAs containing modified backbones or no natural internucleoside linkages. As defined in this specification, dsRNAs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified dsRNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

Preferred modified dsRNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference

Preferred modified dsRNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or ore or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.

In other preferred dsRNA mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an dsRNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an dsRNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are dsRNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAs having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified dsRNAs may also contain one or more substituted sugar moieties. Preferred dsRNAs comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. Other preferred dsRNAs comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an dsRNA, or a group for improving the pharmacodynamic properties of an dsRNA, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other preferred modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the dsRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. DsRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

DsRNAs may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosine's, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.

Conjugates

Another modification of the dsRNAs of the invention involves chemically linking to the dsRNA one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the dsRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 199, 86, 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994 4 1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

Representative U.S. patents that teach the preparation of such dsRNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an dsRNA. The present invention also includes dsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compounds or “chimeras,” in the context of this invention, are dsRNA compounds, particularly dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an dsRNA compound. These dsRNAs typically contain at least one region wherein the dsRNA is modified so as to confer upon the dsRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the dsRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of dsRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter dsRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

In certain instances, the dsRNA may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to dsRNAs in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such dsRNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of dsRNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the dsRNA still bound to the solid support or following cleavage of the dsRNA in solution phase. Purification of the dsRNA conjugate by HPLC typically affords the pure conjugate.

In some cases, a ligand can be multifunctional and/or a dsRNA can be conjugated to more than one ligand. For example, the dsRNA can be conjugated to one ligand for improved uptake and to a second ligand for improved release.

Vector Encoded RNAi Agents

In another aspect of the invention, Eg5 and VEGF specific dsRNA molecules that are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In a preferred embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

The recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129)); adenovirus (see, for example, Berkner, et al., BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. Nati. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA 87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Natl. Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.

Any viral vector capable of accepting the coding sequences for the dsRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.

For example, lentiviral vectors of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors of the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.

Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the dsRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat. Genet. 33: 401-406, the entire disclosures of which are herein incorporated by reference.

Preferred viral vectors are those derived from AV and AAV. In a particularly preferred embodiment, the dsRNA of the invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the dsRNA of the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the dsRNA of the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski Ret al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.

The promoter driving dsRNA expression in either a DNA plasmid or viral vector of the invention may be a eukaryotic RNA polymerase I (e.g. ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter or actin promoter or Ul snRNA promoter) or generally RNA polymerase III promoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter. The promoter can also direct transgene expression to the pancreas (see, e.g., the insulin regulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g. Oligofectamine) or non-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single EG5 gene (or VEGF gene) or multiple Eg5 genes (or VEGF genes) over a period of a week or more are also contemplated by the invention. Successful introduction of the vectors of the invention into host cells can be monitored using various known methods. For example, transient transfection. can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of ex vivo cells can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

The Eg5 specific dsRNA molecules and VEGF specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

Pharmaceutical Compositions Containing dsRNA

In one embodiment, the invention provides pharmaceutical compositions containing a dsRNA, as described herein, and a pharmaceutically acceptable carrier and methods of administering the same. The pharmaceutical composition containing the dsRNA is useful for treating a disease or disorder associated with the expression or activity of a Eg5/KSP and/or VEGF gene, such as pathological processes mediated by Eg5/KSP and/or VEGF expression, e.g., liver cancer. Such pharmaceutical compositions are formulated based on the mode of delivery.

Dosage

The pharmaceutical compositions featured herein are administered in dosages sufficient to inhibit expression of EG5/KSP and/or VEGF genes. In general, a suitable dose of dsRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.01 mg/kg, 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose.

The pharmaceutical composition can be administered once daily, or the dsRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day. The effect of a single dose on EG5/KSP AND/OR VEGF levels is long lasting, such that subsequent doses are administered at not more than 7 day intervals, or at not more than 1, 2, 3, or 4 week intervals.

In some embodiments the dsRNA is administered using continuous infusion or delivery through a controlled release formulation. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.

Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by EG5/KSP AND/OR VEGFexpression. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose. A suitable mouse model is, for example, a mouse containing a plasmid expressing human EG5/KSP AND/OR VEGF. Another suitable mouse model is a transgenic mouse carrying a transgene that expresses human EG5/KSP AND/OR VEGF.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the dsRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by target gene expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

Administration

The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical, pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, and subdermal, oral or parenteral, e.g., subcutaneous.

Typically, when treating a mammal with hyperlipidemia, the dsRNA molecules are administered systemically via parental means. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intraparenchymal, intrathecal or intraventricular, administration. For example, dsRNAs, conjugated or unconjugate or formulated with or without liposomes, can be administered intravenously to a patient. For such, a dsRNA molecule can be formulated into compositions such as sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions in liquid or solid oil bases. Such solutions also can contain buffers, diluents, and other suitable additives. For parenteral, intrathecal, or intraventricular administration, a dsRNA molecule can be formulated into compositions such as sterile aqueous solutions, which also can contain buffers, diluents, and other suitable additives (e.g., penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers). Formulations are described in more detail herein.

The dsRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).

Formulations

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. In one aspect are formulations that target the liver when treating hepatic disorders such as hyperlipidemia.

In addition, dsRNA that target the EG5/KSP AND/OR VEGFgene can be formulated into compositions containing the dsRNA admixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of nucleic acids. For example, a composition containing one or more dsRNA agents that target the Eg5/KSP and/or VEGFgene can contain other therapeutic agents such as other cancer therapeutics or one or more dsRNA compounds that target non-EG5/KSP AND/OR VEGFgenes.

Oral, Parenteral, Topical, and Biologic Formulations

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, U.S. patent publication. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Suitable topical formulations include those in which the dsRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). DsRNAs featured in the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, dsRNAs may be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₁₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference. In addition, dsRNA molecules can be administered to a mammal as biologic or abiologic means as described in, for example, U.S. Pat. No. 6,271,359. Abiologic delivery can be accomplished by a variety of methods including, without limitation, (1) loading liposomes with a dsRNA acid molecule provided herein and (2) complexing a dsRNA molecule with lipids or liposomes to form nucleic acid-lipid or nucleic acid-liposome complexes. The liposome can be composed of cationic and neutral lipids commonly used to transfect cells in vitro. Cationic lipids can complex (e.g., charge-associate) with negatively charged nucleic acids to form liposomes. Examples of cationic liposomes include, without limitation, lipofectin, lipofectamine, lipofectace, and DOTAP. Procedures for forming liposomes are well known in the art. Liposome compositions can be formed, for example, from phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, or dioleoyl phosphatidylethanolamine Numerous lipophilic agents are commercially available, including Lipofectin™ (Invitrogen/Life Technologies, Carlsbad, Calif.) and Effectene™ (Qiagen, Valencia, Calif.). In addition, systemic delivery methods can be optimized using commercially available cationic lipids such as DDAB or DOTAP, each of which can be mixed with a neutral lipid such as DOPE or cholesterol. In some cases, liposomes such as those described by Templeton et al. (Nature Biotechnology, 15: 647-652 (1997)) can be used. In other embodiments, polycations such as polyethyleneimine can be used to achieve delivery in vivo and ex vivo (Boletta et al., J. Am Soc. Nephrol. 7: 1728 (1996)). Additional information regarding the use of liposomes to deliver nucleic acids can be found in U.S. Pat. No. 6,271,359, PCT Publication WO 96/40964 and Morrissey, D. et al. 2005. Nat Biotechnol. 23(8):1002-7.

Biologic delivery can be accomplished by a variety of methods including, without limitation, the use of viral vectors. For example, viral vectors (e.g., adenovirus and herpesvirus vectors) can be used to deliver dsRNA molecules to liver cells. Standard molecular biology techniques can be used to introduce one or more of the dsRNAs provided herein into one of the many different viral vectors previously developed to deliver nucleic acid to cells. These resulting viral vectors can be used to deliver the one or more dsRNAs to cells by, for example, infection.

Characterization of Formulated dsRNAs

Formulations prepared by either the in-line mixing or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total siRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.

Liposomal Formulations

There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun, 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterolipo-lyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(M1), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_(M1), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Lim et al).

Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C_(1215G), that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N. Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N. Y., 1988, p. 285).

SNALPs

In one embodiment, a dsRNA featured in the invention is fully encapsulated in the lipid formulation to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N—(I-(2,3-dioleyloxyl)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, or a mixture thereof. The cationic lipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), a PEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or a PEG-distearyloxypropyl (C]₈). The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

LNP01

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (Formula 1), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-siRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous siRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-siRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

Emulsions

The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N. Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

In one embodiment of the present invention, the compositions of dsRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or dsRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of dsRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of dsRNAs and nucleic acids.

Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the dsRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly dsRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of dsRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carryier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of dsRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of dsRNAs through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of dsRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs.

Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

Carriers

dsRNAs of the present invention can be formulated in a pharmaceutically acceptable carrier or diluent. A “pharmaceutically acceptable carrier” (also referred to herein as an “excipient”) is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle. Pharmaceutically acceptable carriers can be liquid or solid, and can be selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, and other pertinent transport and chemical properties. Typical pharmaceutically acceptable carriers include, by way of example and not limitation: water; saline solution; binding agents (e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, gelatin, or calcium sulfate); lubricants (e.g., starch, polyethylene glycol, or sodium acetate); disintegrates (e.g., starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The co-administration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extra-circulatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is co-administered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Other Components

The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Combination Therapy

In one aspect, a composition of the invention can be used in combination therapy. The term “combination therapy” includes the administration of the subject compounds in further combination with other biologically active ingredients (such as, but not limited to, a second and different antineoplastic agent) and non-drug therapies (such as, but not limited to, surgery or radiation treatment). For instance, the compounds of the invention can be used in combination with other pharmaceutically active compounds, preferably compounds that are able to enhance the effect of the compounds of the invention. The compounds of the invention can be administered simultaneously (as a single preparation or separate preparation) or sequentially to the other drug therapy. In general, a combination therapy envisions administration of two or more drugs during a single cycle or course of therapy.

In one aspect of the invention, the subject compounds may be administered in combination with one or more separate agents that modulate protein kinases involved in various disease states. Examples of such kinases may include, but are not limited to:

serine/threonine specific kinases, receptor tyrosine specific kinases and non-receptor tyrosine specific kinases. Serine/threonine kinases include mitogen activated protein kinases (MAPK), meiosis specific kinase (MEK), RAF and aurora kinase. Examples of receptor kinase families include epidermal growth factor receptor (EGFR) (e.g., HER2/neu, HER3, HER4, ErbB, ErbB2, ErbB3, ErbB4, Xmrk, DER, Let23); fibroblast growth factor (FGF) receptor (e.g. FGF-R1, GFF-R2/BEK/CEK3, FGF-R3/CEK2, FGF-R4/TKF, KGF-R); hepatocyte growth/scatter factor receptor (HGFR) (e.g., MET, RON, SEA, SEX); insulin receptor (e.g. IGFI-R); Eph (e.g. CEKS, CEK8, EBK, ECK, EEK, EHK-I, EHK-2, ELK, EPH, ERK, HEK, MDK2, MDKS, SEK); AxI (e.g. Mer/Nyk, Rse); RET; and platelet-derived growth factor receptor (PDGFR) (e.g. PDGFα-R, PDGβ-R, CSF1-R/FMS, SCF-R/C-KIT, VEGF-R/FLT, NEK/FLK1, FLT3/FLK2/STK-1). Non-receptor tyrosine kinase families include, but are not limited to, BCR-ABL (e.g. p43^(abl), ARG); BTK (e.g. ITK/EMT, TEC); CSK, FAK, FPS, JAK, SRC, BMX, FER, CDK and SYK.

In another aspect of the invention, the subject compounds may be administered in combination with one or more agents that modulate non-kinase biological targets or processes. Such targets include histone deacetylases (HDAC), DNA methyltransferase (DNMT), heat shock proteins (e.g., HSP90), and proteosomes.

In one embodiment, subject compounds may be combined with antineoplastic agents (e.g. small molecules, monoclonal antibodies, antisense RNA, and fusion proteins) that inhibit one or more biological targets such as Zolinza, Tarceva, Iressa, Tykerb, Gleevec, Sutent, Sprycel, Nexavar, Sorafenib, CNF2024, RG108, BMS387032, Affmitak, Avastin, Herceptin, Erbitux, AG24322, PD325901, ZD6474, PD 184322, Obatodax, ABT737 and AEE788. Such combinations may enhance therapeutic efficacy over efficacy achieved by any of the agents alone and may prevent or delay the appearance of resistant mutational variants.

In certain preferred embodiments, the compounds of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents encompass a wide range of therapeutic treatments in the field of oncology. These agents are administered at various stages of the disease for the purposes of shrinking tumors, destroying remaining cancer cells left over after surgery, inducing remission, maintaining remission and/or alleviating symptoms relating to the cancer or its treatment. Examples of such agents include, but are not limited to, alkylating agents such as mustard gas derivatives (Mechlorethamine, cylophosphamide, chlorambucil, melphalan, ifosfamide), ethylenimines (thiotepa, hexamethylmelanine), Alkylsulfonates (Busulfan), Hydrazines and Triazines (Altretamine, Procarbazine, Dacarbazine and Temozolomide), Nitrosoureas (Carmustine, Lomustine and Streptozocin), Ifosfamide and metal salts (Carboplatin, Cisplatin, and Oxaliplatin); plant alkaloids such as Podophyllotoxins (Etoposide and Tenisopide), Taxanes (Paclitaxel and Docetaxel), Vinca alkaloids (Vincristine, Vinblastine, Vindesine and Vinorelbine), and Camptothecan analogs (Irinotecan and Topotecan); anti-tumor antibiotics such as Chromomycins (Dactinomycin and Plicamycin), Anthracyclines (Doxorubicin, Daunorubicin, Epirubicin, Mitoxantrone, Valrubicin and Idarubicin), and miscellaneous antibiotics such as Mitomycin, Actinomycin and Bleomycin; anti-metabolites such as folic acid antagonists (Methotrexate, Pemetrexed, Raltitrexed, Aminopterin), pyrimidine antagonists (5-Fluorouracil, Floxuridine, Cytarabine, Capecitabine, and Gemcitabine), purine antagonists (6-Mercaptopurine and 6-Thioguanine) and adenosine deaminase inhibitors (Cladribine, Fludarabine, Mercaptopurine, Clofarabine, Thioguanine, Nelarabine and Pentostatin); topoisomerase inhibitors such as topoisomerase I inhibitors (Ironotecan, topotecan) and topoisomerase II inhibitors (Amsacrine, etoposide, etoposide phosphate, teniposide); monoclonal antibodies (Alemtuzumab, Gemtuzumab ozogamicin, Rituximab, Trastuzumab, Ibritumomab Tioxetan, Cetuximab, Panitumumab, Tositumomab, Bevacizumab); and miscellaneous anti-neoplasties such as ribonucleotide reductase inhibitors (Hydroxyurea); adrenocortical steroid inhibitor (Mitotane); enzymes (Asparaginase and Pegaspargase); anti-microtubule agents (Estramustine); and retinoids (Bexarotene, Isotretinoin, Tretinoin (ATRA). In certain preferred embodiments, the compounds of the invention are administered in combination with a chemoprotective agent. Chemoprotective agents act to protect the body or minimize the side effects of chemotherapy. Examples of such agents include, but are not limited to, amfostine, mesna, and dexrazoxane.

In one aspect of the invention, the subject compounds are administered in combination with radiation therapy. Radiation is commonly delivered internally (implantation of radioactive material near cancer site) or externally from a machine that employs photon (x-ray or gamma-ray) or particle radiation. Where the combination therapy further comprises radiation treatment, the radiation treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and radiation treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the radiation treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.

It will be appreciated that compounds of the invention can be used in combination with an immunotherapeutic agent. One form of immunotherapy is the generation of an active systemic tumor-specific immune response of host origin by administering a vaccine composition at a site distant from the tumor. Various types of vaccines have been proposed, including isolated tumor-antigen vaccines and anti-idiotype vaccines. Another approach is to use tumor cells from the subject to be treated, or a derivative of such cells (reviewed by Schirrmacher et al. (1995) J. Cancer Res. Clin. Oncol. 121:487). In U.S. Pat. No. 5,484,596, Hanna Jr. et al. claim a method for treating a resectable carcinoma to prevent recurrence or metastases, comprising surgically removing the tumor, dispersing the cells with collagenase, irradiating the cells, and vaccinating the patient with at least three consecutive doses of about 10⁷ cells.

It will be appreciated that the compounds of the invention may advantageously be used in conjunction with one or more adjunctive therapeutic agents. Examples of suitable agents for adjunctive therapy include steroids, such as corticosteroids (amcinonide, betamethasone, betamethasone dipropionate, betamethasone valerate, budesonide, clobetasol, clobetasol acetate, clobetasol butyrate, clobetasol 17-propionate, cortisone, deflazacort, desoximetasone, diflucortolone valerate, dexamethasone, dexamethasone sodium phosphate, desonide, furoate, fluocinonide, fluocinolone acetonide, halcinonide, hydrocortisone, hydrocortisone butyrate, hydrocortisone sodium succinate, hydrocortisone valerate, methyl prednisolone, mometasone, prednicarbate, prednisolone, triamcinolone, triamcinolone acetonide, and halobetasol proprionate); a SHTi agonist, such as a triptan (e.g. sumatriptan or naratriptan); an adenosine Al agonist; an EP ligand; an NMDA modulator, such as a glycine antagonist; a sodium channel blocker (e.g. lamotrigine); a substance P antagonist (e.g. an NKi antagonist); a cannabinoid; acetaminophen or phenacetin; a 5-lipoxygenase inhibitor; a leukotriene receptor antagonist; a DMARD (e.g. methotrexate); gabapentin and related compounds; a tricyclic antidepressant (e.g. amitryptilline); a neurone stabilizing antiepileptic drug; a mono-aminergic uptake inhibitor (e.g. venlafaxine); a matrix metalloproteinase inhibitor; a nitric oxide synthase (NOS) inhibitor, such as an iNOS or an nNOS inhibitor; an inhibitor of the release, or action, of tumour necrosis factor α; an antibody therapy, such as a monoclonal antibody therapy; an antiviral agent, such as a nucleoside inhibitor (e.g. lamivudine) or an immune system modulator (e.g. interferon); an opioid analgesic; a local anaesthetic; a stimulant, including caffeine; an H2-antagonist (e.g. ranitidine); a proton pump inhibitor (e.g. omeprazole); an antacid (e.g. aluminium or magnesium hydroxide; an antiflatulent (e.g. simethicone); a decongestant (e.g. phenylephrine, phenylpropanolamine, pseudoephedrine, oxymetazoline, epinephrine, naphazoline, xylometazoline, propylhexedrine, or levo-desoxyephedrine); an antitussive (e.g. codeine, hydrocodone, carmiphen, carbetapentane, or dextramethorphan); a diuretic; or a sedating or non-sedating antihistamine.

The compounds of the invention can be co-administered with siRNA that target other genes. For example, a compound of the invention can be co-administered with an siRNA targeted to a c-Myc gene. In one example, AD-12115 can be co-administered with a c-Myc siRNA. Examples of c-Myc targeted siRNAs are disclosed in U.S. patent application Ser. No. 12/373,039 which is herein incorporated by reference.

Methods for Treating Diseases Caused by Expression of the Eg5 and VEGF Genes

The invention relates in particular to the use of a composition containing at least two dsRNAs, one targeting an Eg5 gene, and one targeting a VEGF gene, for the treatment of a cancer, such as liver cancer, e.g., for inhibiting tumor growth and tumor metastasis. For example, a composition, such as pharmaceutical composition, may be used for the treatment of solid tumors, like intrahepatic tumors such as may occur in cancers of the liver. A composition containing a dsRNA targeting Eg5 and a dsRNA targeting VEGF may also be used to treat other tumors and cancers, such as breast cancer, lung cancer, head and neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophagus cancer, gastrointestinal cancer, glioma, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, Wilm's tumor, multiple myeloma and for the treatment of skin cancer, like melanoma, for the treatment of lymphomas and blood cancer. The invention further relates to the use of a composition containing an Eg5 dsRNA and a VEGF dsRNA for inhibiting accumulation of ascites fluid and pleural effusion in different types of cancer, e.g., liver cancer, breast cancer, lung cancer, head cancer, neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophagus cancer, gastrointestinal cancer, glioma, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, Wilm's tumor, multiple myeloma, skin cancer, melanoma, lymphomas and blood cancer. Owing to the inhibitory effects on Eg5 and VEGF expression, a composition according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life.

In one embodiment, a patient having a tumor associated with AFP expression, or a tumor secreting AFP, e.g., a hepatoma or teratoma, is treated. In certain embodiments, the patient has a malignant teratoma, an endodermal sinus tumor (yolk sac carcinoma), a neuroblastoma, a hepatoblastoma, a heptocellular carcinoma, testicular cancer or ovarian cancer.

The invention furthermore relates to the use of a dsRNA or a pharmaceutical composition thereof, e.g., for treating cancer or for preventing tumor metastasis, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating cancer and/or for preventing tumor metastasis. Preference is given to a combination with radiation therapy and chemotherapeutic agents, such as cisplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen.

The invention can also be practiced by including with a specific RNAi agent, in combination with another anti-cancer chemotherapeutic agent, such as any conventional chemotherapeutic agent. The combination of a specific binding agent with such other agents can potentiate the chemotherapeutic protocol. Numerous chemotherapeutic protocols will present themselves in the mind of the skilled practitioner as being capable of incorporation into the method of the invention. Any chemotherapeutic agent can be used, including alkylating agents, antimetabolites, hormones and antagonists, radioisotopes, as well as natural products. For example, the compound of the invention can be administered with antibiotics such as doxorubicin and other anthracycline analogs, nitrogen mustards such as cyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, taxol and its natural and synthetic derivatives, and the like. As another example, in the case of mixed tumors, such as adenocarcinoma of the breast, where the tumors include gonadotropin-dependent and gonadotropin-independent cells, the compound can be administered in conjunction with leuprolide or goserelin (synthetic peptide analogs of LH-RH). Other antineoplastic protocols include the use of a tetracycline compound with another treatment modality, e.g., surgery, radiation, etc., also referred to herein as “adjunct antineoplastic modalities.” Thus, the method of the invention can be employed with such conventional regimens with the benefit of reducing side effects and enhancing efficacy.

Methods for Inhibiting Expression of the Eg5 Gene and the VEGF Gene

In yet another aspect, the invention provides a method for inhibiting the expression of the Eg5 gene and the VEGF gene in a mammal. The method includes administering a composition featured in the invention to the mammal such that expression of the target Eg5 gene and the target VEGF gene is silenced.

In one embodiment, a method for inhibiting Eg5 gene expression and VEGF gene expression includes administering a composition containing two different dsRNA molecules, one having a nucleotide sequence that is complementary to at least a part of an RNA transcript of the Eg5 gene and the other having a nucleotide sequence that is complementary to at least a part of an RNA transcript of the VEGF gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In preferred embodiments, the compositions are administered by intravenous infusion or injection.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES Example 1 dsRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

siRNA Synthesis

For screening of dsRNA, single-stranded RNAs were produced by solid phase synthesis on a scale of 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support. RNA and RNA containing 2′-O-methyl nucleotides were generated by solid phase synthesis employing the corresponding phosphoramidites and 2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anion exchange HPLC were carried out according to established procedures. Yields and concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 640B, Beckman Coulter GmbH, UnterschleiBheim, Germany). Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3 minutes and cooled to room temperature over a period of 3-4 hours. The annealed RNA solution was stored at −20° C. until use.

Conjugates

The following is a prophetic description of the synthesis of 3′-cholesterol-conjugated siRNAs (herein referred to as -Chol-3′), an appropriately modified solid support was used for RNA synthesis. The modified solid support was prepared as follows:

Diethyl-2-Azabutane-1,4-Dicarboxylate AA

A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into a stirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole) was added and the mixture was stirred at room temperature until completion of the reaction was ascertained by TLC. After 19 h the solution was partitioned with dichloromethane (3×100 mL). The organic layer was dried with anhydrous sodium sulfate, filtered and evaporated. The residue was distilled to afford AA (28.8 g, 61%).

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionic acid ethyl ester AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved in dichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It was then followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). The solution was brought to room temperature and stirred further for 6 h. Completion of the reaction was ascertained by TLC. The reaction mixture was concentrated under vacuum and ethyl acetate was added to precipitate diisopropyl urea. The suspension was filtered. The filtrate was washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. The combined organic layer was dried over sodium sulfate and concentrated to give the crude product which was purified by column chromatography (50% EtOAC/Hexanes) to yield 11.87 g (88%) of AB.

3-[6-Amino-Hexanoyl)-Ethoxycarbonylmethyl-Amino]-Propionic Acid Ethyl Ester AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionic acid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidine in dimethylformamide at 0° C. The solution was continued stirring for 1 h. The reaction mixture was concentrated under vacuum, water was added to the residue, and the product was extracted with ethyl acetate. The crude product was purified by conversion into its hydrochloride salt.

3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD

The hydrochloride salt of 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. The suspension was cooled to 0° C. on ice. To the suspension diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To the resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) was added. The reaction mixture was stirred overnight. The reaction mixture was diluted with dichloromethane and washed with 10% hydrochloric acid. The product was purified by flash chromatography (10.3 g, 92%).

1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a] phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylic acid ethyl ester AE

Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of dry toluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) of diester AD was added slowly with stirring within 20 mins. The temperature was kept below 5° C. during the addition. The stirring was continued for 30 mins at 0° C. and 1 mL of glacial acetic acid was added, immediately followed by 4 g of NaH₂PO₄.H₂O in 40 mL of water The resultant mixture was extracted twice with 100 mL of dichloromethane each and the combined organic extracts were washed twice with 10 mL of phosphate buffer each, dried, and evaporated to dryness. The residue was dissolved in 60 mL of toluene, cooled to 0° C. and extracted with three 50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extracts were adjusted to pH 3 with phosphoric acid, and extracted with five 40 mL portions of chloroform which were combined, dried and evaporated to dryness. The residue was purified by column chromatography using 25% ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).

[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AF

Methanol (2 mL) was added dropwise over a period of 1 h to a refluxing mixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued at reflux temperature for 1 h. After cooling to room temperature, 1 N HCl (12.5 mL) was added, the mixture was extracted with ethylacetate (3×40 mL). The combined ethylacetate layer was dried over anhydrous sodium sulfate and concentrated under vacuum to yield the product which was purified by column chromatography (10% MeOH/CHCl₃) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AG

Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5 mL) in vacuo. Anhydrous pyridine (10 mL) and 4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added with stirring. The reaction was carried out at room temperature overnight. The reaction was quenched by the addition of methanol. The reaction mixture was concentrated under vacuum and to the residue dichloromethane (50 mL) was added. The organic layer was washed with 1M aqueous sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residual pyridine was removed by evaporating with toluene. The crude product was purified by column chromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl₃) (1.75 g, 95%).

Succinic acid mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) ester AH

Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (40 mL) and washed with ice cold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was used as such for the next step.

Cholesterol Derivatised CPG AI

Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture of dichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL), 2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in acetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using a wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) was added. The suspension was agitated for 2 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The achieved loading of the CPG was measured by taking UV measurement (37 mM/g).

The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamide group (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivative group (herein referred to as “5′-Chol-”) was performed as described in WO 2004/065601, except that, for the cholesteryl derivative, the oxidation step was performed using the Beaucage reagent in order to introduce a phosphorothioate linkage at the 5′-end of the nucleic acid oligomer.

dsRNA Targeting the Eg5 Gene

Initial Screening Set

siRNA design was carried out to identify siRNAs targeting Eg5 (also known as KIF11, HSKP, KNSL1 and TRIPS). Human mRNA sequences to Eg5, RefSeq ID number:NM_(—)004523, was used.

siRNA duplexes cross-reactive to human and mouse Eg5 were designed. Twenty-four duplexes were synthesized for screening. (Table 1a). A second screening set was defined with 266 siRNAs targeting human Eg5, as well as its rhesus monkey ortholog (Table 2a). An expanded screening set was selected with 328 siRNA targeting human Eg5, with no necessity to hit any Eg5 mRNA of other species (Table 3a).

The sequences for human and a partial rhesus Eg5 mRNAs were downloaded from NCBI Nucleotide database and the human sequence was further on used as reference sequence (Human EG5:NM_(—)004523.2, 4908 bp, and Rhesus EG5: XM_(—) 001087644.1, 878 by (only 5′ part of human EG5)

For the Tables: Key: A,G,C,U-ribonucleotides: T-deoxythymidine: u,c-2′-O-methyl nucleotides: s-phosphorothioate linkage.

TABLE 1a Sequences of Eg5/KSP dsRNA duplexes position  in human SEQ sequence of SEQ SEQ Eg5/KSP ID 23mer target ID sense sequence ID antisense sequence duplex sequence NO: site NO: (5′-3′) No: (5′-3′) name  385-407 1244 ACCGAAGUGUUGUUUGUC  1 cGAAGuGuuGuuuGuccA  2 UUGGAcAAAcAAcACUUCG AL-DP- CAAUU ATsT TsT 6226  347-369 1245 UAUGGUGUUUGGAGCAUC  3 uGGuGuuuGGAGcAucuA  4 GuAGAUGCUCcAAAcACcA AL-DP- UACUA cTsT TsT 6227 1078-1100 1246 AAUCUAAACUAACUAGAA  5 ucuAAAcuAAcuAGAAuc  6 GGAUUCuAGUuAGUUuAGA AL-DP- UCCUC cTsT TsT 6228 1067-1089 1247 UCCUUAUCGAGAAUCUAA  7 cuuAucGAGAAucuAAAc  8 AGUUuAGAUUCUCGAuAAG AL-DP- ACUAA uTsT TsT 6229  374-396 1248 GAUUGAUGUUUACCGAAG  9 uuGAuGuuuAccGAAGuG 10 AcACUUCGGuAAAcAUcAA AL-DP- UGUUG uTsT TsT 6230  205-227 1249 UGGUGAGAUGCAGACCAU 11 GuGAGAuGcAGAccAuuu 12 uAAAUGGUCUGcAUCUcAC AL-DP- UUAAU ATsT TsT 6231 1176-1198 1250 ACUCUGAGUACAUUGGAA 13 ucuGAGuAcAuuGGAAuA 14 AuAUUCcAAUGuACUcAGA AL-DP- UAUGC uTsT TsT 6232  386-408 1251 CCGAAGUGUUGUUUGUCC 15 GAAGuGuuGuuuGuccAA 16 AUUGGAcAAAcAAcACUUC AL-DP- AAUUC uTsT TsT 6233  416-438 1252 AGUUAUUAUGGGCUAUAA 17 uuAuuAuGGGcuAuAAuu 18 cAAUuAuAGCCcAuAAuAA AL-DP- UUGCA GTsT TsT 6234  485-507 1253 GGAAGGUGAAAGGUCACC 19 AAGGuGAAAGGucAccuA 20 UuAGGUGACCUUUcACCUU AL-DP- UAAUG ATsT TsT 6235  476-498 1254 UUUUACAAUGGAAGGUGA 21 uuAcAAuGGAAGGuGAAA 22 CUUUcACCUUCcAUUGuAA AL-DP- AAGGU GTsT TsT 6236  486-508 1255 GAAGGUGAAAGGUCACCU 23 AGGuGAAAGGucAccuAA 24 AUuAGGUGACCUUUcACCU AL-DP- AAUGA uTsT TsT 6237  487-509 1256 AAGGUGAAAGGUCACCUA 25 GGuGAAAGGucAccuAAu 26 cAUuAGGUGACCUUUcACC AL-DP- AUGAA GTsT TsT 6238 1066-1088 1257 UUCCUUAUCGAGAAUCUA 27 ccuuAucGAGAAucuAAA 28 GUUuAGAUUCUCGAuAAGG AL-DP- AACUA cTsT TsT 6239 1256-1278 1258 AGCUCUUAUUAAGGAGUA 29 cucuuAuuAAGGAGuAuA 30 GuAuACUCCUuAAuAAGAG AL-DP- UACGG cTsT TsT 6240 2329-2351 1259 CAGAGAGAUUCUGUGCUU 31 GAGAGAuucuGuGcuuuG 32 CcAAAGcAcAGAAUCUCUC AL-DP- UGGAG GTsT TsT 6241 1077-1099 1260 GAAUCUAAACUAACUAGA 33 AucuAAAcuAAcuAGAAu 34 GAUUCuAGUuAGUUuAGAU AL-DP- AUCCU cTsT TsT 6242 1244-1266 1261 ACUCACCAAAAAAGCUCU 35 ucAccAAAAAAGcucuuA 36 AuAAGAGCUUUUUUGGUGA AL-DP- UAUUA uTsT TsT 6243  637-659 1262 AAGAGCUUUUUGAUCUUC 37 GAGcuuuuuGAucuucuu 38 uAAGAAGAUcAAAAAGCUC AL-DP- UUAAU ATsT TsT 6244 1117-1139 1263 GGCGUACAAGAACAUCUA 39 cGuAcAAGAAcAucuAuA 40 UuAuAGAUGUUCUUGuACG AL-DP- UAAUU ATsT TsT 6245  373-395  126 AGAUUGAUGUUUACCGAA 41 AuuGAuGuuuAccGAAGu 42 cACUUCGGuAAAcAUcAAU AL-DP-    4 GUGUU GTsT TsT 6246 1079-  126 AUCUAAACUAACUAGAAU 43 cuAAAcuAAcuAGAAucc 44 AGGAUUCuAGUuAGUUuAG AL-DP- 1101    5 CCUCC uTsT TsT 6247  383-405  126 UUACCGAAGUGUUGUUUG 45 AccGAAGuGuuGuuuGuc 46 GGAcAAAcAAcACUUCGGU AL-DP-    6 UCCAA cTsT TsT 6248  200-222  126 GGUGGUGGUGAGAUGCAG 47 uGGuGGuGAGAuGcAGAc 48 GGUCUGcAUCUcACcACcA AL-DP-    7 ACCAU cTsT TsT 6249

TABLE 1b Analysis of Eg5/KSP ds duplexes single dose screen @ 25 nM [% SDs 2nd screen duplex residual (among name mRNA] quadruplicates) AL-DP-6226 23% 3% AL-DP-6227 69% 10%  AL-DP-6228 33% 2% AL-DP-6229  2% 2% AL-DP-6230 66% 11%  AL-DP-6231 17% 1% AL-DP-6232  9% 3% AL-DP-6233 24% 6% AL-DP-6234 91% 2% AL-DP-6235 112%  4% AL-DP-6236 69% 4% AL-DP-6237 42% 2% AL-DP-6238 45% 2% AL-DP-6239  2% 1% AL-DP-6240 48% 2% AL-DP-6241 41% 2% AL-DP-6242  8% 2% AL-DP-6243  7% 1% AL-DP-6244  6% 2% AL-DP-6245 12% 2% AL-DP-6246 28% 3% AL-DP-6247 71% 4% AL-DP-6248  5% 2% AL-DP-6249 28% 3%

TABLE 2a Sequences of Eg5/KSP dsRNA duplexes SEQ SEQ SEQ ID sequence of 19-mer ID sense sequence ID antisense sequence duplex NO: target site NO. (5′-3′) NO. (5′-3′) name 1268 CAUACUCUAGUCGUUCCCA  49 cAuAcucuAGucGuucccATsT  50 UGGGAACGACuAGAGuAUGTsT AD-12072 1269 AGCGCCCAUUCAAUAGUAG  51 AGcGcccAuucAAuAGuAGTsT  52 CuACuAUUGAAUGGGCGCUTsT AD-12073 1270 GGAAAGCUAGCGCCCAUUC  53 GGAAAGcuAGcGcccAuucTsT  54 GAAUGGGCGCuAGCUUUCCTsT AD-12074 1271 GAAAGCUAGCGCCCAUUCA  55 GAAAGcuAGcGcccAuucATsT  56 UGAAUGGGCGCuAGCUUUCTsT AD-12075 1272 AGAAACUACGAUUGAUGGA  57 AGAAAcuAcGAuuGAuGGATsT  58 UCcAUcAAUCGuAGUUUCUTsT AD-12076 1273 UGUUCCUUAUCGAGAAUCU  59 uGuuccuuAucGAGAAucuTsT  60 AGAUUCUCGAuAAGGAAcATsT AD-12077 1274 CAGAUUACCUCUGCGAGCC  61 cAGAuuAccucuGcGAGccTsT  62 GGCUCGcAGAGGuAAUCUGTsT AD-12078 1275 GCGCCCAUUCAAUAGUAGA  63 GcGcccAuucAAuAGuAGATsT  64 UCuACuAUUGAAUGGGCGCTsT AD-12079 1276 UUGCACUAUCUUUGCGUAU  65 uuGcAcuAucuuuGcGuAuTsT  66 AuACGcAAAGAuAGUGcAATsT AD-12080 1277 CAGAGCGGAAAGCUAGCGC  67 cAGAGcGGAAAGcuAGcGcTsT  68 GCGCuAGCUUUCCGCUCUGTsT AD-12081 1278 AGACCUUAUUUGGUAAUCU  69 AGAccuuAuuuGGuAAucuTsT  70 AGAUuACcAAAuAAGGUCUTsT AD-12082 1279 AUUCUCUUGGAGGGCGUAC  71 AuucucuuGGAGGGcGuAcTsT  72 GuACGCCCUCcAAGAGAAUTsT AD-12083 1280 GGCUGGUAUAAUUCCACGU  73 GGcuGGuAuAAuuccAcGuTsT  74 ACGUGGAAUuAuACcAGCCTsT AD-12084 1281 GCGGAAAGCUAGCGCCCAU  75 GcGGAAAGcuAGcGcccAuTsT  76 AUGGGCGCuAGCUUUCCGCTsT AD-12085 1282 UGCACUAUCUUUGCGUAUG  77 uGcAcuAucuuuGcGuAuGTsT  78 cAuACGcAAAGAuAGUGcATsT AD-12086 1283 GUAUAAUUCCACGUACCCU  79 GuAuAAuuccAcGuAcccuTsT  80 AGGGuACGUGGAAUuAuACTsT AD-12087 1284 AGAAUCUAAACUAACUAGA  81 AGAAucuAAAcuAAcuAGATsT  82 UCuAGUuAGUUuAGAUUCUTsT AD-12088 1285 AGGAGCUGAAUAGGGUUAC  83 AGGAGcuGAAuAGGGuuAcTsT  84 GuAACCCuAUUcAGCUCCUTsT AD-12089 1286 GAAGUACAUAAGACCUUAU  85 GAAGuAcAuAAGAccuuAuTsT  86 AuAAGGUCUuAUGuACUUCTsT AD-12090 1287 GACAGUGGCCGAUAAGAUA  87 GAcAGuGGccGAuAAGAuATsT  88 uAUCUuAUCGGCcACUGUCTsT AD-12091 1288 AAACCACUUAGUAGUGUCC  89 AAAccAcuuAGuAGuGuccTsT  90 GGAcACuACuAAGUGGUUUTsT AD-12092 1289 UCCCUAGACUUCCCUAUUU  91 ucccuAGAcuucccuAuuuTsT  92 AAAuAGGGAAGUCuAGGGATsT AD-12093 1290 UAGACUUCCCUAUUUCGCU  93 uAGAcuucccuAuuucGcuTsT  94 AGCGAAAuAGGGAAGUCuATsT AD-12094 1291 GCGUCGCAGCCAAAUUCGU  95 GcGucGcAGccAAAuucGuTsT  96 ACGAAUUUGGCUGCGACGCTsT AD-12095 1292 AGCUAGCGCCCAUUCAAUA  97 AGcuAGcGcccAuucAAuATsT  98 uAUUGAAUGGGCGCuAGCUTsT AD-12096 1293 GAAACUACGAUUGAUGGAG  99 GAAAcuAcGAuuGAuGGAGTsT 100 CUCcAUcAAUCGuAGUUUCTsT AD-12097 1294 CCGAUAAGAUAGAAGAUCA 101 ccGAuAAGAuAGAAGAucATsT 102 UGAUCUUCuAUCUuAUCGGTsT AD-12098 1295 UAGCGCCCAUUCAAUAGUA 103 uAGcGcccAuucAAuAGuATsT 104 uACuAUUGAAUGGGCGCuATsT AD-12099 1296 UUUGCGUAUGGCCAAACUG 105 uuuGcGuAuGGccAAAcuGTsT 106 cAGUUUGGCcAuACGcAAATsT AD-12100 1297 CACGUACCCUUCAUCAAAU 107 cAcGuAcccuucAucAAAuTsT 108 AUUUGAUGAAGGGuACGUGTsT AD-12101 1298 UCUUUGCGUAUGGCCAAAC 109 ucuuuGcGuAuGGccAAAcTsT 110 GUUUGGCcAuACGcAAAGATsT AD-12102 1299 CCGAAGUGUUGUUUGUCCA 111 ccGAAGuGuuGuuuGuccATsT 112 UGGAcAAAcAAcACUUCGGTsT AD-12103 1300 AGAGCGGAAAGCUAGCGCC 113 AGAGcGGAAAGcuAGcGccTsT 114 GGCGCuAGCUUUCCGCUCUTsT AD-12104 1301 GCUAGCGCCCAUUCAAUAG 115 GcuAGcGcccAuucAAuAGTsT 116 CuAUUGAAUGGGCGCuAGCTsT AD-12105 1302 AAGUUAGUGUACGAACUGG 117 AAGuuAGuGuAcGAAcuGGTsT 118 CcAGUUCGuAcACuAACUUTsT AD-12106 1303 GUACGAACUGGAGGAUUGG 119 GuAcGAAcuGGAGGAuuGGTsT 120 CcAAUCCUCcAGUUCGuACTsT AD-12107 1304 ACGAACUGGAGGAUUGGCU 121 AcGAAcuGGAGGAuuGGcuTsT 122 AGCcAAUCCUCcAGUUCGUTsT AD-12108 1305 AGAUUGAUGUUUACCGAAG 123 AGAuuGAuGuuuAccGAAGTsT 124 CUUCGGuAAAcAUcAAUCUTsT AD-12109 1306 UAUGGGCUAUAAUUGCACU 125 uAuGGGcuAuAAuuGcAcuTsT 126 AGUGcAAUuAuAGCCcAuATsT AD-12110 1307 AUCUUUGCGUAUGGCCAAA 127 AucuuuGcGuAuGGccAAATsT 128 UUUGGCcAuACGcAAAGAUTsT AD-12111 1308 ACUCUAGUCGUUCCCACUC 129 AcucuAGucGuucccAcucTsT 130 GAGUGGGAACGACuAGAGUTsT AD-12112 1309 AACUACGAUUGAUGGAGAA 131 AAcuAcGAuuGAuGGAGAATsT 132 UUCUCcAUcAAUCGuAGUUTsT AD-12113 1310 GAUAAGAGAGCUCGGGAAG 133 GAuAAGAGAGcucGGGAAGTsT 134 CUUCCCGAGCUCUCUuAUCTsT AD-12114 1311 UCGAGAAUCUAAACUAACU 135 ucGAGAAucuAAAcuAAcuTsT 136 AGUuAGUUuAGAUUCUCGATsT AD-12115 1312 AACUAACUAGAAUCCUCCA 137 AAcuAAcuAGAAuccuccATsT 138 UGGAGGAUUCuAGUuAGUUTsT AD-12116 1313 GGAUCGUAAGAAGGCAGUU 139 GGAucGuAAGAAGGcAGuuTsT 140 AACUGCCUUCUuACGAUCCTsT AD-12117 1314 AUCGUAAGAAGGCAGUUGA 141 AucGuAAGAAGGcAGuuGATsT 142 UcAACUGCCUUCUuACGAUTsT AD-12118 1315 AGGCAGUUGACCAACACAA 143 AGGcAGuuGAccAAcAcAATsT 144 UUGUGUUGGUcAACUGCCUTsT AD-12119 1316 UGGCCGAUAAGAUAGAAGA 145 uGGccGAuAAGAuAGAAGATsT 146 UCUUCuAUCUuAUCGGCcATsT AD-12120 1317 UCUAAGGAUAUAGUCAACA 147 ucuAAGGAuAuAGucAAcATsT 148 UGUUGACuAuAUCCUuAGATsT AD-12121 1318 ACUAAGCUUAAUUGCUUUC 149 AcuAAGcuuAAuuGcuuucTsT 150 GAAAGcAAUuAAGCUuAGUTsT AD-12122 1319 GCCCAGAUCAACCUUUAAU 151 GcccAGAucAAccuuuAAuTsT 152 AUuAAAGGUUGAUCUGGGCTsT AD-12123 1320 UUAAUUUGGCAGAGCGGAA 153 uuAAuuuGGcAGAGcGGAATsT 154 UUCCGCUCUGCcAAAUuAATsT AD-12124 1321 UUAUCGAGAAUCUAAACUA 155 uuAucGAGAAucuAAAcuATsT 156 uAGUUuAGAUUCUCGAuAATsT AD-12125 1322 CUAGCGCCCAUUCAAUAGU 157 cuAGcGcccAuucAAuAGuTsT 158 ACuAUUGAAUGGGCGCuAGTsT AD-12126 1323 AAUAGUAGAAUGUGAUCCU 159 AAuAGuAGAAuGuGAuccuTsT 160 AGGAUcAcAUUCuACuAUUTsT AD-12127 1324 UACGAAAAGAAGUUAGUGU 161 uAcGAAAAGAAGuuAGuGuTsT 162 AcACuAACUUCUUUUCGuATsT AD-12128 1325 AGAAGUUAGUGUACGAACU 163 AGAAGuuAGuGuAcGAAcuTsT 164 AGUUCGuAcACuAACUUCUTsT AD-12129 1326 ACUAAACAGAUUGAUGUUU 165 AcuAAAcAGAuuGAuGuuuTsT 166 AAAcAUcAAUCUGUUuAGUTsT AD-12130 1327 CUUUGCGUAUGGCCAAACU 167 cuuuGcGuAuGGccAAAcuTsT 168 AGUUUGGCcAuACGcAAAGTsT AD-12131 1328 AAUGAAGAGUAUACCUGGG 169 AAuGAAGAGuAuAccuGGGTsT 170 CCcAGGuAuACUCUUcAUUTsT AD-12132 1329 AUAAUUCCACGUACCCUUC 171 AuAAuuccAcGuAcccuucTsT 172 GAAGGGuACGUGGAAUuAUTsT AD-12133 1330 ACGUACCCUUCAUCAAAUU 173 AcGuAcccuucAucAAAuuTsT 174 AAUUUGAUGAAGGGuACGUTsT AD-12134 1331 CGUACCCUUCAUCAAAUUU 175 cGuAcccuucAucAAAuuuTsT 176 AAAUUUGAUGAAGGGuACGTsT AD-12135 1332 GUACCCUUCAUCAAAUUUU 177 GuAcccuucAucAAAuuuuTsT 178 AAAAUUUGAUGAAGGGuACTsT AD-12136 1333 AACUUACUGAUAAUGGUAC 179 AAcuuAcuGAuAAuGGuAcTsT 180 GuACcAUuAUcAGuAAGUUTsT AD-12137 1334 UUCAGUCAAAGUGUCUCUG 181 uucAGucAAAGuGucucuGTsT 182 cAGAGAcACUUUGACUGAATsT AD-12138 1335 UUCUUAAUCCAUCAUCUGA 183 uucuuAAuccAucAucuGATsT 184 UcAGAUGAUGGAUuAAGAATsT AD-12139 1336 ACAGUACACAACAAGGAUG 185 AcAGuAcAcAAcAAGGAuGTsT 186 cAUCCUUGUUGUGuACUGUTsT AD-12140 1337 AAGAAACUACGAUUGAUGG 187 AAGAAAcuAcGAuuGAuGGTsT 188 CcAUcAAUCGuAGUUUCUUTsT AD-12141 1338 AAACUACGAUUGAUGGAGA 189 AAAcuAcGAuuGAuGGAGATsT 190 UCUCcAUcAAUCGuAGUUUTsT AD-12142 1339 UGGAGCUGUUGAUAAGAGA 191 uGGAGcuGuuGAuAAGAGATsT 192 UCUCUuAUcAAcAGCUCcATsT AD-12143 1340 CUAACUAGAAUCCUCCAGG 193 cuAAcuAGAAuccuccAGGTsT 194 CCUGGAGGAUUCuAGUuAGTsT AD-12144 1341 GAAUAUGCUCAUAGAGCAA 195 GAAuAuGcucAuAGAGcAATsT 196 UUGCUCuAUGAGcAuAUUCTsT AD-12145 1342 AUGCUCAUAGAGCAAAGAA 197 AuGcucAuAGAGcAAAGAATsT 198 UUCUUUGCUCuAUGAGcAUTsT AD-12146 1343 AAAAAUUGGUGCUGUUGAG 199 AAAAAuuGGuGcuGuuGAGTsT 200 CUcAAcAGcACcAAUUUUUTsT AD-12147 1344 GAGGAGCUGAAUAGGGUUA 201 GAGGAGcuGAAuAGGGuuATsT 202 uAACCCuAUUcAGCUCCUCTsT AD-12148 1345 GGAGCUGAAUAGGGUUACA 203 GGAGcuGAAuAGGGuuAcATsT 204 UGuAACCCuAUUcAGCUCCTsT AD-12149 1346 GAGCUGAAUAGGGUUACAG 205 GAGcuGAAuAGGGuuAcAGTsT 206 CUGuAACCCuAUUcAGCUCTsT AD-12150 1347 AGCUGAAUAGGGUUACAGA 207 AGcuGAAuAGGGuuAcAGATsT 208 UCUGuAACCCuAUUcAGCUTsT AD-12151 1348 GCUGAAUAGGGUUACAGAG 209 GcuGAAuAGGGuuAcAGAGTsT 210 CUCUGuAACCCuAUUcAGCTsT AD-12152 1349 CCAAACUGGAUCGUAAGAA 211 ccAAAcuGGAucGuAAGAATsT 212 UUCUuACGAUCcAGUUUGGTsT AD-12153 1350 GAUCGUAAGAAGGCAGUUG 213 GAucGuAAGAAGGcAGuuGTsT 214 cAACUGCCUUCUuACGAUCTsT AD-12154 1351 ACCUUAUUUGGUAAUCUGC 215 AccuuAuuuGGuAAucuGcTsT 216 GcAGAUuACcAAAuAAGGUTsT AD-12155 1352 UUAGAUACCAUUACUACAG 217 uuAGAuAccAuuAcuAcAGTsT 218 CUGuAGuAAUGGuAUCuAATsT AD-12156 1353 AUACCAUUACUACAGUAGC 219 AuAccAuuAcuAcAGuAGcTsT 220 GCuACUGuAGuAAUGGuAUTsT AD-12157 1354 UACUACAGUAGCACUUGGA 221 uAcuAcAGuAGcAcuuGGATsT 222 UCcAAGUGCuACUGuAGuATsT AD-12158 1355 AAAGUAAAACUGUACUACA 223 AAAGuAAAAcuGuAcuAcATsT 224 UGuAGuAcAGUUUuACUUUTsT AD-12159 1356 CUCAAGACUGAUCUUCUAA 225 cucAAGAcuGAucuucuAATsT 226 UuAGAAGAUcAGUCUUGAGTsT AD-12160 1357 UUGACAGUGGCCGAUAAGA 227 uuGAcAGuGGccGAuAAGATsT 228 UCUuAUCGGCcACUGUcAATsT AD-12161 1358 UGACAGUGGCCGAUAAGAU 229 uGAcAGuGGccGAuAAGAuTsT 230 AUCUuAUCGGCcACUGUcATsT AD-12162 1359 GCAAUGUGGAAACCUAACU 231 GcAAuGuGGAAAccuAAcuTsT 232 AGUuAGGUUUCcAcAUUGCTsT AD-12163 1360 CCACUUAGUAGUGUCCAGG 233 ccAcuuAGuAGuGuccAGGTsT 234 CCUGGAcACuACuAAGUGGTsT AD-12164 1361 AGAAGGUACAAAAUUGGUU 235 AGAAGGuAcAAAAuuGGuuTsT 236 AACcAAUUUUGuACCUUCUTsT AD-12165 1362 UGGUUUGACUAAGCUUAAU 237 uGGuuuGAcuAAGcuuAAuTsT 238 AUuAAGCUuAGUcAAACcATsT AD-12166 1363 GGUUUGACUAAGCUUAAUU 239 GGuuuGAcuAAGcuuAAuuTsT 240 AAUuAAGCUuAGUcAAACCTsT AD-12167 1364 UCUAAGUCAAGAGCCAUCU 241 ucuAAGucAAGAGccAucuTsT 242 AGAUGGCUCUUGACUuAGATsT AD-12168 1365 UCAUCCCUAUAGUUCACUU 243 ucAucccuAuAGuucAcuuTsT 244 AAGUGAACuAuAGGGAUGATsT AD-12169 1366 CAUCCCUAUAGUUCACUUU 245 cAucccuAuAGuucAcuuuTsT 246 AAAGUGAACuAuAGGGAUGTsT AD-12170 1367 CCCUAGACUUCCCUAUUUC 247 cccuAGAcuucccuAuuucTsT 248 GAAAuAGGGAAGUCuAGGGTsT AD-12171 1368 AGACUUCCCUAUUUCGCUU 249 AGAcuucccuAuuucGcuuTsT 250 AAGCGAAAuAGGGAAGUCUTsT AD-12172 1369 UCACCAAACCAUUUGUAGA 251 ucAccAAAccAuuuGuAGATsT 252 UCuAcAAAUGGUUUGGUGATsT AD-12173 1370 UCCUUUAAGAGGCCUAACU 253 uccuuuAAGAGGccuAAcuTsT 254 AGUuAGGCCUCUuAAAGGATsT AD-12174 1371 UUUAAGAGGCCUAACUCAU 255 uuuAAGAGGccuAAcucAuTsT 256 AUGAGUuAGGCCUCUuAAATsT AD-12175 1372 UUAAGAGGCCUAACUCAUU 257 uuAAGAGGccuAAcucAuuTsT 258 AAUGAGUuAGGCCUCUuAATsT AD-12176 1373 GGCCUAACUCAUUCACCCU 259 GGccuAAcucAuucAcccuTsT 260 AGGGUGAAUGAGUuAGGCCTsT AD-12177 1374 UGGUAUUUUUGAUCUGGCA 261 uGGuAuuuuuGAucuGGcATsT 262 UGCcAGAUcAAAAAuACcATsT AD-12178 1375 AGUUUAGUGUGUAAAGUUU 263 AGuuuAGuGuGuAAAGuuuTsT 264 AAACUUuAcAcACuAAACUTsT AD-12179 1376 GCCAAAUUCGUCUGCGAAG 265 GccAAAuucGucuGcGAAGTsT 266 CUUCGcAGACGAAUUUGGCTsT AD-12180 1377 AAUUCGUCUGCGAAGAAGA 267 AAuucGucuGcGAAGAAGATsT 268 UCUUCUUCGcAGACGAAUUTsT AD-12181 1378 UGAAAGGUCACCUAAUGAA 269 uGAAAGGucAccuAAuGAATsT 270 UUcAUuAGGUGACCUUUcATsT AD-12182 1379 CAGACCAUUUAAUUUGGCA 271 cAGAccAuuuAAuuuGGcATsT 272 UGCcAAAUuAAAUGGUCUGTsT AD-12183 1380 AGACCAUUUAAUUUGGCAG 273 AGAccAuuuAAuuuGGcAGTsT 274 CUGCcAAAUuAAAUGGUCUTsT AD-12184 1381 AGUUAUUAUGGGCUAUAAU 275 AGuuAuuAuGGGcuAuAAuTsT 276 AUuAuAGCCcAuAAuAACUTsT AD-12185 1382 GCUGGUAUAAUUCCACGUA 277 GcuGGuAuAAuuccAcGuATsT 278 uACGUGGAAUuAuACcAGCTsT AD-12186 1383 AUUUAAUUUGGCAGAGCGG 279 AuuuAAuuuGGcAGAGcGGTsT 280 CCGCUCUGCcAAAUuAAAUTsT AD-12187 1384 UUUAAUUUGGCAGAGCGGA 281 uuuAAuuuGGcAGAGcGGATsT 282 UCCGCUCUGCcAAAUuAAATsT AD-12188 1385 UUUGGCAGAGCGGAAAGCU 283 uuuGGcAGAGcGGAAAGcuTsT 284 AGCUUUCCGCUCUGCcAAATsT AD-12189 1386 UUUUACAAUGGAAGGUGAA 285 uuuuAcAAuGGAAGGuGAATsT 286 UUcACCUUCcAUUGuAAAATsT AD-12190 1387 AAUGGAAGGUGAAAGGUCA 287 AAuGGAAGGuGAAAGGucATsT 288 UGACCUUUcACCUUCcAUUTsT AD-12191 1388 UGAGAUGCAGACCAUUUAA 289 uGAGAuGcAGAccAuuuAATsT 290 UuAAAUGGUCUGcAUCUcATsT AD-12192 1389 UCGCAGCCAAAUUCGUCUG 291 ucGcAGccAAAuucGucuGTsT 292 cAGACGAAUUUGGCUGCGATsT AD-12193 1390 GGCUAUAAUUGCACUAUCU 293 GGcuAuAAuuGcAcuAucuTsT 294 AGAuAGUGcAAUuAuAGCCTsT AD-12194 1391 AUUGACAGUGGCCGAUAAG 295 AuuGAcAGuGGccGAuAAGTsT 296 CUuAUCGGCcACUGUcAAUTsT AD-12195 1392 CUAGACUUCCCUAUUUCGC 297 cuAGAcuucccuAuuucGcTsT 298 GCGAAAuAGGGAAGUCuAGTsT AD-12196 1393 ACUAUCUUUGCGUAUGGCC 299 AcuAucuuuGcGuAuGGccTsT 300 GGCcAuACGcAAAGAuAGUTsT AD-12197 1394 AUACUCUAGUCGUUCCCAC 301 AuAcucuAGucGuucccAcTsT 302 GUGGGAACGACuAGAGuAUTsT AD-12198 1395 AAAGAAACUACGAUUGAUG 303 AAAGAAAcuAcGAuuGAuGTsT 304 cAUcAAUCGuAGUUUCUUUTsT AD-12199 1396 GCCUUGAUUUUUUGGCGGG 305 GccuuGAuuuuuuGGcGGGTsT 306 CCCGCcAAAAAAUcAAGGCTsT AD-12200 1397 CGCCCAUUCAAUAGUAGAA 307 cGcccAuucAAuAGuAGAATsT 308 UUCuACuAUUGAAUGGGCGTsT AD-12201 1398 CCUUAUUUGGUAAUCUGCU 309 ccuuAuuuGGuAAucuGcuTsT 310 AGcAGAUuACcAAAuAAGGTsT AD-12202 1399 AGAGACAAUUCCGGAUGUG 311 AGAGAcAAuuccGGAuGuGTsT 312 cAcAUCCGGAAUUGUCUCUTsT AD-12203 1400 UGACUUUGAUAGCUAAAUU 313 uGAcuuuGAuAGcuAAAuuTsT 314 AAUUuAGCuAUcAAAGUcATsT AD-12204 1401 UGGCAGAGCGGAAAGCUAG 315 uGGcAGAGcGGAAAGcuAGTsT 316 CuAGCUUUCCGCUCUGCcATsT AD-12205 1402 GAGCGGAAAGCUAGCGCCC 317 GAGcGGAAAGcuAGcGcccTsT 318 GGGCGCuAGCUUUCCGCUCTsT AD-12206 1403 AAAGAAGUUAGUGUACGAA 319 AAAGAAGuuAGuGuAcGAATsT 320 UUCGuAcACuAACUUCUUUTsT AD-12207 1404 AUUGCACUAUCUUUGCGUA 321 AuuGcAcuAucuuuGcGuATsT 322 uACGcAAAGAuAGUGcAAUTsT AD-12208 1405 GGUAUAAUUCCACGUACCC 323 GGuAuAAuuccAcGuAcccTsT 324 GGGuACGUGGAAUuAuACCTsT AD-12209 1406 UACUCUAGUCGUUCCCACU 325 uAcucuAGucGuucccAcuTsT 326 AGUGGGAACGACuAGAGuATsT AD-12210 1407 UAUGAAAGAAACUACGAUU 327 uAuGAAAGAAAcuAcGAuuTsT 328 AAUCGuAGUUUCUUUcAuATsT AD-12211 1408 AUGCUAGAAGUACAUAAGA 329 AuGcuAGAAGuAcAuAAGATsT 330 UCUuAUGuACUUCuAGcAUTsT AD-12212 1409 AAGUACAUAAGACCUUAUU 331 AAGuAcAuAAGAccuuAuuTsT 332 AAuAAGGUCUuAUGuACUUTsT AD-12213 1410 ACAGCCUGAGCUGUUAAUG 333 AcAGccuGAGcuGuuAAuGTsT 334 cAUuAAcAGCUcAGGCUGUTsT AD-12214 1411 AAAGAAGAGACAAUUCCGG 335 AAAGAAGAGAcAAuuccGGTsT 336 CCGGAAUUGUCUCUUCUUUTsT AD-12215 1412 CACACUGGAGAGGUCUAAA 337 cAcAcuGGAGAGGucuAAATsT 338 UUuAGACCUCUCcAGUGUGTsT AD-12216 1413 CACUGGAGAGGUCUAAAGU 339 cAcuGGAGAGGucuAAAGuTsT 340 ACUUuAGACCUCUCcAGUGTsT AD-12217 1414 ACUGGAGAGGUCUAAAGUG 341 AcuGGAGAGGucuAAAGuGTsT 342 cACUUuAGACCUCUCcAGUTsT AD-12218 1415 CGUCGCAGCCAAAUUCGUC 343 cGucGcAGccAAAuucGucTsT 344 GACGAAUUUGGCUGCGACGTsT AD-12219 1416 GAAGGCAGUUGACCAACAC 345 GAAGGcAGuuGAccAAcAcTsT 346 GUGUUGGUcAACUGCCUUCTsT AD-12220 1417 CAUUCACCCUGACAGAGUU 347 cAuucAcccuGAcAGAGuuTsT 348 AACUCUGUcAGGGUGAAUGTsT AD-12221 1418 AAGAGGCCUAACUCAUUCA 349 AAGAGGccuAAcucAuucATsT 350 UGAAUGAGUuAGGCCUCUUTsT AD-12222 1419 GAGACAAUUCCGGAUGUGG 351 GAGAcAAuuccGGAuGuGGTsT 352 CcAcAUCCGGAAUUGUCUCTsT AD-12223 1420 UUCCGGAUGUGGAUGUAGA 353 uuccGGAuGuGGAuGuAGATsT 354 UCuAcAUCcAcAUCCGGAATsT AD-12224 1421 AAGCUAGCGCCCAUUCAAU 355 AAGcuAGcGcccAuucAAuTsT 356 AUUGAAUGGGCGCuAGCUUTsT AD-12225 1422 GAAGUUAGUGUACGAACUG 357 GAAGuuAGuGuAcGAAcuGTsT 358 cAGUUCGuAcACuAACUUCTsT AD-12226 1423 UAUAAUUCCACGUACCCUU 359 uAuAAuuccAcGuAcccuuTsT 360 AAGGGuACGUGGAAUuAuATsT AD-12227 1424 ACAGUGGCCGAUAAGAUAG 361 AcAGuGGccGAuAAGAuAGTsT 362 CuAUCUuAUCGGCcACUGUTsT AD-12228 1425 UCUGUCAUCCCUAUAGUUC 363 ucuGucAucccuAuAGuucTsT 364 GAACuAuAGGGAUGAcAGATsT AD-12229 1426 UUCUUGCUAUGACUUGUGU 365 uucuuGcuAuGAcuuGuGuTsT 366 AcAcAAGUcAuAGcAAGAATsT AD-12230 1427 GUAAGAAGGCAGUUGACCA 367 GuAAGAAGGcAGuuGAccATsT 368 UGGUcAACUGCCUUCUuACTsT AD-12231 1428 CAUUGACAGUGGCCGAUAA 369 cAuuGAcAGuGGccGAuAATsT 370 UuAUCGGCcACUGUcAAUGTsT AD-12232 1429 AGAAACCACUUAGUAGUGU 371 AGAAAccAcuuAGuAGuGuTsT 372 AcACuACuAAGUGGUUUCUTsT AD-12233 1430 GGAUUGUUCAUCAAUUGGC 373 GGAuuGuucAucAAuuGGcTsT 374 GCcAAUUGAUGAAcAAUCCTsT AD-12234 1431 UAAGAGGCCUAACUCAUUC 375 uAAGAGGccuAAcucAuucTsT 376 GAAUGAGUuAGGCCUCUuATsT AD-12235 1432 AGUUAGUGUACGAACUGGA 377 AGuuAGuGuAcGAAcuGGATsT 378 UCcAGUUCGuAcACuAACUTsT AD-12236 1433 AGUACAUAAGACCUUAUUU 379 AGuAcAuAAGAccuuAuuuTsT 380 AAAuAAGGUCUuAUGuACUTsT AD-12237 1434 UGAGCCUUGUGUAUAGAUU 381 uGAGccuuGuGuAuAGAuuTsT 382 AAUCuAuAcAcAAGGCUcATsT AD-12238 1435 CCUUUAAGAGGCCUAACUC 383 ccuuuAAGAGGccuAAcucTsT 384 GAGUuAGGCCUCUuAAAGGTsT AD-12239 1436 ACCACUUAGUAGUGUCCAG 385 AccAcuuAGuAGuGuccAGTsT 386 CUGGAcACuACuAAGUGGUTsT AD-12240 1437 GAAACUUCCAAUUAUGUCU 387 GAAAcuuccAAuuAuGucuTsT 388 AGAcAuAAUUGGAAGUUUCTsT AD-12241 1438 UGCAUACUCUAGUCGUUCC 389 uGcAuAcucuAGucGuuccTsT 390 GGAACGACuAGAGuAUGcATsT AD-12242 1439 AGAAGGCAGUUGACCAACA 391 AGAAGGcAGuuGAccAAcATsT 392 UGUUGGUcAACUGCCUUCUTsT AD-12243 1440 GUACAUAAGACCUUAUUUG 393 GuAcAuAAGAccuuAuuuGTsT 394 cAAAuAAGGUCUuAUGuACTsT AD-12244 1441 UAUAAUUGCACUAUCUUUG 395 uAuAAuuGcAcuAucuuuGTsT 396 cAAAGAuAGUGcAAUuAuATsT AD-12245 1442 UCUCUGUUACAAUACAUAU 397 ucucuGuuAcAAuAcAuAuTsT 398 AuAUGuAUUGuAAcAGAGATsT AD-12246 1443 UAUGCUCAUAGAGCAAAGA 399 uAuGcucAuAGAGcAAAGATsT 400 UCUUUGCUCuAUGAGcAuATsT AD-12247 1444 UGUUGUUUGUCCAAUUCUG 401 uGuuGuuuGuccAAuucuGTsT 402 cAGAAUUGGAcAAAcAAcATsT AD-12248 1445 ACUAACUAGAAUCCUCCAG 403 AcuAAcuAGAAuccuccAGTsT 404 CUGGAGGAUUCuAGUuAGUTsT AD-12249 1446 UGUGGUGUCUAUACUGAAA 405 uGuGGuGucuAuAcuGAAATsT 406 UUUcAGuAuAGAcACcAcATsT AD-12250 1447 UAUUAUGGGAGACCACCCA 407 uAuuAuGGGAGAccAcccATsT 408 UGGGUGGUCUCCcAuAAuATsT AD-12251 1448 AAGGAUGAAGUCUAUCAAA 409 AAGGAuGAAGucuAucAAATsT 410 UUUGAuAGACUUcAUCCUUTsT AD-12252 1449 UUGAUAAGAGAGCUCGGGA 411 uuGAuAAGAGAGcucGGGATsT 412 UCCCGAGCUCUCUuAUcAATsT AD-12253 1450 AUGUUCCUUAUCGAGAAUC 413 AuGuuccuuAucGAGAAucTsT 414 GAUUCUCGAuAAGGAAcAUTsT AD-12254 1451 GGAAUAUGCUCAUAGAGCA 415 GGAAuAuGcucAuAGAGcATsT 416 UGCUCuAUGAGcAuAUUCCTsT AD-12255 1452 CCAUUCCAAACUGGAUCGU 417 ccAuuccAAAcuGGAucGuTsT 418 ACGAUCcAGUUUGGAAUGGTsT AD-12256 1453 GGCAGUUGACCAACACAAU 419 GGcAGuuGAccAAcAcAAuTsT 420 AUUGUGUUGGUcAACUGCCTsT AD-12257 1454 CAUGCUAGAAGUACAUAAG 421 cAuGcuAGAAGuAcAuAAGTsT 422 CUuAUGuACUUCuAGcAUGTsT AD-12258 1455 CUAGAAGUACAUAAGACCU 423 cuAGAAGuAcAuAAGAccuTsT 424 AGGUCUuAUGuACUUCuAGTsT AD-12259 1456 UUGGAUCUCUCACAUCUAU 425 uuGGAucucucAcAucuAuTsT 426 AuAGAUGUGAGAGAUCcAATsT AD-12260 1457 AACUGUGGUGUCUAUACUG 427 AAcuGuGGuGucuAuAcuGTsT 428 cAGuAuAGAcACcAcAGUUTsT AD-12261 1458 UCAUUGACAGUGGCCGAUA 429 ucAuuGAcAGuGGccGAuATsT 430 uAUCGGCcACUGUcAAUGATsT AD-12262 1459 AUAAAGCAGACCCAUUCCC 431 AuAAAGcAGAcccAuucccTsT 432 GGGAAUGGGUCUGCUUuAUTsT AD-12263 1460 ACAGAAACCACUUAGUAGU 433 AcAGAAAccAcuuAGuAGuTsT 434 ACuACuAAGUGGUUUCUGUTsT AD-12264 1461 GAAACCACUUAGUAGUGUC 435 GAAAccAcuuAGuAGuGucTsT 436 GAcACuACuAAGUGGUUUCTsT AD-12265 1462 AAAUCUAAGGAUAUAGUCA 437 AAAucuAAGGAuAuAGucATsT 438 UGACuAuAUCCUuAGAUUUTsT AD-12266 1463 UUAUUUAUACCCAUCAACA 439 uuAuuuAuAcccAucAAcATsT 440 UGUUGAUGGGuAuAAAuAATsT AD-12267 1464 ACAGAGGCAUUAACACACU 441 AcAGAGGcAuuAAcAcAcuTsT 442 AGUGUGUuAAUGCCUCUGUTsT AD-12268 1465 ACACACUGGAGAGGUCUAA 443 AcAcAcuGGAGAGGucuAATsT 444 UuAGACCUCUCcAGUGUGUTsT AD-12269 1466 ACACUGGAGAGGUCUAAAG 445 AcAcuGGAGAGGucuAAAGTsT 446 CUUuAGACCUCUCcAGUGUTsT AD-12270 1467 CGAGCCCAGAUCAACCUUU 447 cGAGcccAGAucAAccuuuTsT 448 AAAGGUUGAUCUGGGCUCGTsT AD-12271 1468 UCCCUAUUUCGCUUUCUCC 449 ucccuAuuucGcuuucuccTsT 450 GGAGAAAGCGAAAuAGGGATsT AD-12272 1469 UCUAAAAUCACUGUCAACA 451 ucuAAAAucAcuGucAAcATsT 452 UGUUGAcAGUGAUUUuAGATsT AD-12273 1470 AGCCAAAUUCGUCUGCGAA 453 AGccAAAuucGucuGcGAATsT 454 UUCGcAGACGAAUUUGGCUTsT AD-12274 1471 CCCAUUCAAUAGUAGAAUG 455 cccAuucAAuAGuAGAAuGTsT 456 cAUUCuACuAUUGAAUGGGTsT AD-12275 1472 GAUGAAUGCAUACUCUAGU 457 GAuGAAuGcAuAcucuAGuTsT 458 ACuAGAGuAUGcAUUcAUCTsT AD-12276 1473 CUCAUGUUCCUUAUCGAGA 459 cucAuGuuccuuAucGAGATsT 460 UCUCGAuAAGGAAcAUGAGTsT AD-12277 1474 GAGAAUCUAAACUAACUAG 461 GAGAAucuAAAcuAAcuAGTsT 462 CuAGUuAGUUuAGAUUCUCTsT AD-12278 1475 UAGAAGUACAUAAGACCUU 463 uAGAAGuAcAuAAGAccuuTsT 464 AAGGUCUuAUGuACUUCuATsT AD-12279 1476 CAGCCUGAGCUGUUAAUGA 465 cAGccuGAGcuGuuAAuGATsT 466 UcAUuAAcAGCUcAGGCUGTsT AD-12280 1477 AAGAAGAGACAAUUCCGGA 467 AAGAAGAGAcAAuuccGGATsT 468 UCCGGAAUUGUCUCUUCUUTsT AD-12281 1478 UGCUGGUGUGGAUUGUUCA 469 uGcuGGuGuGGAuuGuucATsT 470 UGAAcAAUCcAcACcAGcATsT AD-12282 1479 AAAUUCGUCUGCGAAGAAG 471 AAAuucGucuGcGAAGAAGTsT 472 CUUCUUCGcAGACGAAUUUTsT AD-12283 1480 UUUCUGGAAGUUGAGAUGU 473 uuucuGGAAGuuGAGAuGuTsT 474 AcAUCUcAACUUCcAGAAATsT AD-12284 1481 UACUAAACAGAUUGAUGUU 475 uAcuAAAcAGAuuGAuGuuTsT 476 AAcAUcAAUCUGUMIAGuATsT AD-12285 1482 GAUUGAUGUUUACCGAAGU 477 GAuuGAuGuuuAccGAAGuTsT 478 ACUUCGGuAAAcAUcAAUCTsT AD-12286 1483 GCACUAUCUUUGCGUAUGG 479 GcAcuAucuuuGcGuAuGGTsT 480 CcAuACGcAAAGAuAGUGCTsT AD-12287 1484 UGGUAUAAUUCCACGUACC 481 uGGuAuAAuuccAcGuAccTsT 482 GGuACGUGGAAUuAuACcATsT AD-12288 1485 AGCAAGCUGCUUAACACAG 483 AGcAAGcuGcuuAAcAcAGTsT 484 CUGUGUuAAGcAGCUUGCUTsT AD-12289 1486 CAGAAACCACUUAGUAGUG 485 cAGAAAccAcuuAGuAGuGTsT 486 cACuACuAAGUGGUUUCUGTsT AD-12290 1487 AACUUAUUGGAGGUUGUAA 487 AAcuuAuuGGAGGuuGuAATsT 488 UuAcAACCUCcAAuAAGUUTsT AD-12291 1488 CUGGAGAGGUCUAAAGUGG 489 cuGGAGAGGucuAAAGuGGTsT 490 CcACUUuAGACCUCUCcAGTsT AD-12292 1489 AAAAAAGAUAUAAGGCAGU 491 AAAAAAGAuAuAAGGcAGuTsT 492 ACUGCCUuAuAUCUUUUUUTsT AD-12293 1490 GAAUUUUGAUAUCUACCCA 493 GAAuuuuGAuAucuAcccATsT 494 UGGGuAGAuAUcAAAAUUCTsT AD-12294 1491 GUAUUUUUGAUCUGGCAAC 495 GuAuuuuuGAucuGGcAAcTsT 496 GUUGCcAGAUcAAAAAuACTsT AD-12295 1492 AGGAUCCCUUGGCUGGUAU 497 AGGAucccuuGGcuGGuAuTsT 498 AuACcAGCcAAGGGAUCCUTsT AD-12296 1493 GGAUCCCUUGGCUGGUAUA 499 GGAucccuuGGcuGGuAuATsT 500 uAuACcAGCcAAGGGAUCCTsT AD-12297 1494 CAAUAGUAGAAUGUGAUCC 501 cAAuAGuAGAAuGuGAuccTsT 502 GGAUcAcAUUCuACuAUUGTsT AD-12298 1495 GCUAUAAUUGCACUAUCUU 503 GcuAuAAuuGcAcuAucuuTsT 504 AAGAuAGUGcAAUuAuAGCTsT AD-12299 1496 UACCCUUCAUCAAAUUUUU 505 uAcccuucAucAAAuuuuuTsT 506 AAAAAUUUGAUGAAGGGuATsT AD-12300 1497 AGAACAUAUUGAAUAAGCC 507 AGAAcAuAuuGAAuAAGccTsT 508 GGCUuAUUcAAuAUGUUCUTsT AD-12301 1498 AAAUUGGUGCUGUUGAGGA 509 AAAuuGGuGcuGuuGAGGATsT 510 UCCUcAAcAGcACcAAUUUTsT AD-12302 1499 UGAAUAGGGUUACAGAGUU 511 uGAAuAGGGuuAcAGAGuuTsT 512 AACUCUGuAACCCuAUUcATsT AD-12303 1500 AAGAACUUGAAACCACUCA 513 AAGAAcuuGAAAccAcucATsT 514 UGAGUGGUUUcAAGUUCUUTsT AD-12304 1501 AAUAAAGCAGACCCAUUCC 515 AAuAAAGcAGAcccAuuccTsT 516 GGAAUGGGUCUGCUUuAUUTsT AD-12305 1502 AUACCCAUCAACACUGGUA 517 AuAcccAucAAcAcuGGuATsT 518 uACcAGUGUUGAUGGGuAUTsT AD-12306 1503 UGGAUUGUUCAUCAAUUGG 519 uGGAuuGuucAucAAuuGGTsT 520 CcAAUUGAUGAAcAAUCcATsT AD-12307 1504 UGGAGAGGUCUAAAGUGGA 521 uGGAGAGGucuAAAGuGGATsT 522 UCcACUUuAGACCUCUCcATsT AD-12308 1505 GUCAUCCCUAUAGUUCACU 523 GucAucccuAuAGuucAcuTsT 524 AGUGAACuAuAGGGAUGACTsT AD-12309 1506 AUAAUGGCUAUAAUUUCUC 525 AuAAuGGcuAuAAuuucucTsT 526 GAGAAAUuAuAGCcAUuAUTsT AD-12310 1507 AUCCCUUGGCUGGUAUAAU 527 AucccuuGGcuGGuAuAAuTsT 528 AUuAuACcAGCcAAGGGAUTsT AD-12311 1508 GGGCUAUAAUUGCACUAUC 529 GGGcuAuAAuuGcAcuAucTsT 530 GAuAGUGcAAUuAuAGCCCTsT AD-12312 1509 GAUUCUCUUGGAGGGCGUA 531 GAuucucuuGGAGGGcGuATsT 532 uACGCCCUCcAAGAGAAUCTsT AD-12313 1510 GCAUCUCUCAAUCUUGAGG 533 GcAucucucAAucuuGAGGTsT 534 CCUcAAGAUUGAGAGAUGCTsT AD-12314 1511 CAGCAGAAAUCUAAGGAUA 535 cAGcAGAAAucuAAGGAuATsT 536 uAUCCUuAGAUUUCUGCUGTsT AD-12315 1512 GUCAAGAGCCAUCUGUAGA 537 GucAAGAGccAucuGuAGATsT 538 UCuAcAGAUGGCUCUUGACTsT AD-12316 1513 AAACAGAGGCAUUAACACA 539 AAAcAGAGGcAuuAAcAcATsT 540 UGUGUuAAUGCCUCUGUUUTsT AD-12317 1514 AGCCCAGAUCAACCUUUAA 541 AGcccAGAucAAccuuuAATsT 542 UuAAAGGUUGAUCUGGGCUTsT AD-12318 1515 UAUUUUUGAUCUGGCAACC 543 uAuuuuuGAucuGGcAAccTsT 544 GGUUGCcAGAUcAAAAAuATsT AD-12319 1516 UGUUUGGAGCAUCUACUAA 545 uGuuuGGAGcAucuAcuAATsT 546 UuAGuAGAUGCUCcAAAcATsT AD-12320 1517 GAAAUUACAGUACACAACA 547 GAAAuuAcAGuAcAcAAcATsT 548 UGUUGUGuACUGuAAUUUCTsT AD-12321 1518 ACUUGACCAGUGUAAAUCU 549 AcuuGAccAGuGuAAAucuTsT 550 AGAUUuAcACUGGUcAAGUTsT AD-12322 1519 ACCAGUGUAAAUCUGACCU 551 AccAGuGuAAAucuGAccuTsT 552 AGGUcAGAUUuAcACUGGUTsT AD-12323 1520 AGAACAAUCAUUAGCAGCA 553 AGAAcAAucAuuAGcAGcATsT 554 UGCUGCuAAUGAUUGUUCUTsT AD-12324 1521 CAAUGUGGAAACCUAACUG 555 cAAuGuGGAAAccuAAcuGTsT 556 cAGUuAGGUUUCcAcAUUGTsT AD-12325 1522 ACCAAGAAGGUACAAAAUU 557 AccAAGAAGGuAcAAAAuuTsT 558 AAUUUUGuACCUUCUUGGUTsT AD-12326 1523 GGUACAAAAUUGGUUGAAG 559 GGuAcAAAAuuGGuuGAAGTsT 560 CUUcAACcAAUUUUGuACCTsT AD-12327 1524 GGUGUGGAUUGUUCAUCAA 561 GGuGuGGAuuGuucAucAATsT 562 UUGAUGAAcAAUCcAcACCTsT AD-12328 1525 AGAGUUCACAAAAAGCCCA 563 AGAGuucAcAAAAAGcccATsT 564 UGGGCUUUUUGUGAACUCUTsT AD-12329 1526 UGAUAGCUAAAUUAAACCA 565 uGAuAGcuAAAuuAAAccATsT 566 UGGUUuAAUUuAGCuAUcATsT AD-12330 1527 AAUAAGCCUGAAGUGAAUC 567 AAuAAGccuGAAGuGAAucTsT 568 GAUUcACUUcAGGCUuAUUTsT AD-12331 1528 CAGUUGACCAACACAAUGC 569 cAGuuGAccAAcAcAAuGcTsT 570 GcAUUGUGUUGGUcAACUGTsT AD-12332 1529 UGGUGUGGAUUGUUCAUCA 571 uGGuGuGGAuuGuucAucATsT 572 UGAUGAAcAAUCcAcACcATsT AD-12333 1530 AUUCACCCUGACAGAGUUC 573 AuucAcccuGAcAGAGuucTsT 574 GAACUCUGUcAGGGUGAAUTsT AD-12334 1531 UAAGACCUUAUUUGGUAAU 575 uAAGAccuuAuuuGGuAAuTsT 576 AUuACcAAAuAAGGUCUuATsT AD-12335 1532 AAGCAAUGUGGAAACCUAA 577 AAGcAAuGuGGAAAccuAATsT 578 UuAGGUUUCcAcAUUGCUUTsT AD-12336 1533 UCUGAAACUGGAUAUCCCA 579 ucuGAAAcuGGAuAucccATsT 580 UGGGAuAUCcAGUUUcAGATsT AD-12337

TABLE 2b Analysis of Eg5/KSP dsRNA duplexes 1st single SDs 1st 2nd single SDs 2nd SDs 3^(rd) dose screen screen dose screen screen screen Eg5/KSP @ 50 nM (among @ 25 nM (among 3rd single (among duplex [% resudual quadru- [% resudual quadru- dose screen quadru- name mRNA] plicates) mRNA] plicates) @ 25 nM plicates) AD-12072 65% 2% 82% 5% AD-12073 84% 1% 61% 6% AD-12074 51% 3% 36% 9% AD-12075 56% 4% 36% 4% AD-12076 21% 4% 13% 3% AD-12077 11% 2%  6% 1% AD-12078 22% 3%  9% 2% AD-12079 22% 10%  15% 7% AD-12080 68% 4% 52% 13%  AD-12081 34% 8% 35% 24%  AD-12082 20% 2% 92% 5% AD-12083 85% 6% 63% 10%  AD-12084 18% 6% 17% 4% AD-12085 13% 4% 12% 4% AD-12086 26% 5% 17% 3% AD-12087 95% 4% 80% 4% AD-12088 29% 6% 29% 2% AD-12089 69% 5% 64% 7% AD-12090 46% 15%  34% 5% AD-12091 16% 6% 17% 3% AD-12092 82% 26%  63% 5% AD-12093 84% 4% 70% 4% AD-12094 46% 3% 34% 1% AD-12095 14% 2% 13% 1% AD-12096 26% 11%  17% 1% AD-12097 23% 2% 21% 1% AD-12098 41% 14%  17% 3% AD-12099 57% 2% 48% 6% AD-12100 101%  11%  98% 8% AD-12101 46% 7% 32% 2% AD-12102 96% 17%  88% 18%  AD-12103 19% 5% 20% 2% AD-12104 40% 8% 24% 2% AD-12105 39% 2% 36% 10%  AD-12106 87% 6% 79% 19%  AD-12107 29% 2% 32% 16%  AD-12108 38% 4% 39% 8% AD-12109 49% 3% 44% 10%  AD-12110 85% 5% 80% 14%  AD-12111 64% 6% 71% 18%  AD-12112 48% 4% 41% 5% AD-12113 13% 0% 14% 3% AD-12114 32% 6% 16% 4% AD-12115  8% 4%  7% 5% AD-12116 74% 5% 61% 7% AD-12117 21% 4% 20% 2% AD-12118 44% 4% 42% 6% AD-12119 37% 4% 24% 3% AD-12120 22% 2% 15% 4% AD-12121 32% 1% 22% 2% AD-12122 36% 16%  19% 5% AD-12123 28% 1% 16% AD-12124 28% 2% 16% AD-12125 15% 1% 14% AD-12126 51% 22%  27% AD-12127 54% 4% 42% 9% AD-12128 29% 1% 20% 2% AD-12129 22% 3% 19% 3% AD-12130 53% 6% 42% 7% AD-12131 28% 5% 22% 3% AD-12132 88% 2% 90% 18%  AD-12133 34% 2% 26% 6% AD-12134 18% 3% 14% 2% AD-12135 50% 6% 37% 4% AD-12136 42% 19%  22% 2% AD-12137 85% 12%  92% 4% AD-12138 47% 6% 49% 1% AD-12139 80% 5% 72% 4% AD-12140 97% 22%  67% 9% AD-12141 120%  4% 107%  10%  AD-12142 55% 8% 33% 4% AD-12143 64% 34%  19% 2% AD-12144 58% 29%  17% 2% AD-12145 27% 8% 18% 2% AD-12146 19% 20%  15% 1% AD-12147 29% 9% 35% 3% AD-12148 30% 3% 56% 5% AD-12149  8% 2% 12% 3% AD-12150 31% 2% 31% 7% AD-12151  9% 5% 14% 2% AD-12152  3% 3% 23% 3% AD-12153 20% 6% 34% 4% AD-12154 24% 7% 44% 3% AD-12155 33% 6% 53% 11%  AD-12156 35% 5% 40% 5% AD-12157  8% 3% 23% 4% AD-12158 13% 2% 22% 5% AD-12159 34% 6% 46% 5% AD-12160 19% 3% 31% 4% AD-12161 88% 4% 83% 7% AD-12162 26% 7% 32% 7% AD-12163 55% 9% 40% 3% AD-12164 21% 3% AD-12165 30% 3% 41% 4% AD-12166  9% 10%  22% 9% AD-12167 26% 3% 30% 2% AD-12168 54% 4% 59% 20%  AD-12169 41% 4% 51% 16%  AD-12170 43% 4% 52% 20%  AD-12171 67% 3% 73% 25%  AD-12172 53% 15%  37% 2% AD-12173 39% 0% 39% 0% AD-12174 41% 5% 27% 0% AD-12175 29% 0% 38% 14%  AD-12176 43% 2% 56% 25%  AD-12177 68% 6% 74% 30%  AD-12178 41% 4% 41% 6% AD-12179 53% 5% 44% 5% AD-12180 16% 2% 13% 4% AD-12181 19% 3% 14% 2% AD-12182 16% 4% 18% 8% AD-12183 26% 3% 19% 4% AD-12184 54% 2% 77% 8% AD-12185  8% 1%  9% 1% AD-12186 36% 3% 41% 6% AD-12187 34% 17%  27% 1% AD-12188 30% 3% 27% 4% AD-12189 51% 4% 48% 5% AD-12190 33% 2% 26% 4% AD-12191 20% 2% 13% 0% AD-12192 21% 1% 23% 10%  AD-12193 64% 8% 98% 6% AD-12194  8% 2% 15% 4% AD-12195 34% 2% 48% 3% AD-12196 34% 2% 51% 3% AD-12197 75% 4% 93% 6% AD-12198 55% 5% 48% 2% AD-12199 102%  6% 118%  9% AD-12200 75% 6% 60% 12%  AD-12201 42% 3% 16% 4% AD-12202 29% 4%  9% 3% AD-12203 114%  14%  89% 20%  AD-12204 64% 7% 26% 5% AD-12205 66% 12%  35% 4% AD-12206 46% 3% 32% 12%  AD-12207 57% 5% 40% 6% AD-12208 30% 8% 10% 5% AD-12209 101%  6% 102%  23%  AD-12210 38% 11%  27% 14%  AD-12211 16% 6% 10% 5% AD-12212 59% 8% 65% 5% AD-12213 24% 9% 12% 2% AD-12214 67% 14%  70% 12%  AD-12215 29% 13%  13% 4% AD-12216 36% 4% 13% 1% AD-12217 36% 9% 11% 2% AD-12218 35% 5% 17% 3% AD-12219 41% 9% 14% 1% AD-12220 37% 5% 23% 3% AD-12221 58% 7% 39% 6% AD-12222 74% 9% 53% 3% AD-12223 74% 10%  67% 7% AD-12224 24% 2% 11% 2% AD-12225 75% 5% 76% 14%  AD-12226 45% 8% 40% 3% AD-12227 61% 6% 47% 5% AD-12228 28% 3% 25% 5% AD-12229 54% 13%  37% 6% AD-12230 70% 17%  65% 4% AD-12231 32% 12%  22% 6% AD-12232 30% 3% 17% 2% AD-12233 38% 2% 32% 3% AD-12234 90% 5% 95% 7% AD-12235 57% 7% 46% 3% AD-12236 34% 8% 16% 2% AD-12237 42% 9% 32% 8% AD-12238 42% 6% 34% 6% AD-12239 42% 3% 40% 4% AD-12240 47% 6% 36% 5% AD-12241 69% 5% 70% 8% AD-12242 61% 2% 47% 3% AD-12243 26% 7% 15% 1% AD-12244 25% 6% 15% 1% AD-12245 65% 6% 83% 13%  AD-12246 29% 7% 31% 6% AD-12247 57% 13%  50% 3% AD-12248 36% 8% 20% 3% 15% 7% AD-12249 44% 3% 70% 11%  103%  34%  AD-12250 47% 5% 18% 5% 17% 4% AD-12251 121%  28%  35% 8% 60% 42%  AD-12252 94% 19%   8% 3%  5% 3% AD-12253 94% 33%  42% 8% 49% 27%  AD-12254 101%  58%  70% 5% 80% 32%  AD-12255 163%  27%  28% 6% 36% 10%  AD-12256 112%  62%  18% 3%  9% 4% AD-12257 10% 4%  9% 2%  6% 2% AD-12258 27% 9% 18% 3% 20% 6% AD-12259 20% 5% 12% 2% 13% 5% AD-12260 22% 7% 81% 7% 65% 13%  AD-12261 122%  11%  66% 7% 80% 22%  AD-12262 97% 30%  33% 6% 44% 18%  AD-12263 177%  57%  85% 11%  84% 15%  AD-12264 37% 6% 10% 1% 10% 4% AD-12265 40% 8% 17% 1% 20% 10%  AD-12266 33% 9%  9% 1%  8% 4% AD-12267 34% 13%  11% 1%  6% 2% AD-12268 34% 6% 11% 1%  9% 2% AD-12269 54% 6% 33% 4% 29% 7% AD-12270 52% 5% 29% 4% 27% 6% AD-12271 53% 7% 27% 3% 19% 6% AD-12272 85% 15%  57% 7% 51% 16%  AD-12273 36% 6% 26% 2% 30% 5% AD-12274 75% 21%  40% 2% 50% 19%  AD-12275 29% 9%  8% 1%  8% 4% AD-12276 45% 19%  15% 2% 16% 12%  AD-12277 58% 17%  32% 2% 55% 14%  AD-12278 120%  35%  96% 10%  124%  38%  AD-12279 47% 29%  17% 1% 12% 4% AD-12280  2% 0%  3% 1% AD-12281  2% 0%  5% 2% AD-12282  3% 0% 25% 5% AD-12283  3% 1% 35% 4% AD-12284  5% 2% 49% 8% AD-12285  7% 7% 21% 26%  AD-12286 28% 34%  12% 7% AD-12287 40% 21%  51% 23%  AD-12288 26% 7% 155%  146%  AD-12289 43% 21%  220%  131%  AD-12290  2% 1% 81% 23%  AD-12291  4% 1% 70% 3% AD-12292  2% 1%  6% 2% AD-12293  4% 2% 36% 3% AD-12294 10% 6% 38% 3% AD-12295 29% 31%  37% 3% AD-12296 82% 4% 89% 2% AD-12297 75% 3% 65% 2% AD-12298 73% 4% 60% 3% AD-12299 76% 4% 66% 4% AD-12300 36% 4% 15% 1% AD-12301 33% 4% 18% 2% AD-12302 66% 5% 65% 3% AD-12303 35% 6% 17% 2% AD-12304 70% 8% 70% 6% AD-12305 63% 8% 80% 7% AD-12306 23% 6% 20% 3% AD-12307 78% 10%  58% 5% AD-12308 27% 8% 15% 2% AD-12309 58% 11%  42% 3% AD-12310 106%  23%  80% 2% AD-12311 73% 12%  60% 2% AD-12312 39% 3% 36% 3% AD-12313 64% 9% 49% 6% AD-12314 28% 7% 14% 6% AD-12315 31% 7% 13% 2% AD-12316 42% 5% 14% 2% AD-12317 34% 9% 15% 5% AD-12318 46% 4% 28% 4% AD-12319 77% 3% 56% 4% AD-12320 55% 7% 41% 3% AD-12321 21% 3% 10% 2% AD-12322 27% 8% 30% 12%  AD-12323 26% 7% 35% 18%  AD-12324 27% 8% 27% 14%  AD-12325 32% 12%  32% 22%  AD-12326 42% 22%  45% 41%  AD-12327 36% 14%  37% 32%  AD-12328 45% 2% 31% 3% AD-12329 61% 4% 34% 3% AD-12330 63% 5% 38% 4% AD-12331 50% 2% 26% 5% AD-12332 80% 4% 51% 7% AD-12333 34% 6% 12% 2% AD-12334 27% 2% 18% 3% AD-12335 84% 6% 60% 7% AD-12336 45% 4% 36% 4% AD-12337 30% 7% 19% 2%

TABLE 3 Sequences and analysis of Eg5/KSP dsRNA duplexes single SDs 2nd dose screen screen @ (among SEQ SEQ 25 nM [% quadru- Sense sequence ID Antisense sequence ID duplex residual pli- (5′-3′) NO. (5′-3′) NO. name mRNA] cates) ccAuuAcuAcAGuAGcAcuTsT  582 AGUGCuACUGuAGuAAUGGTsT  583 AD-14085  19%  1% AucuGGcAAccAuAuuucuTsT  584 AGAAAuAUGGUUGCcAGAUTsT  585 AD-14086  38%  1% GAuAGcuAAAuuAAAccAATsT  586 UUGGUUuAAUUuAGCuAUCTsT  587 AD-14087  75% 10% AGAuAccAuuAcuAcAGuATsT  588 uACUGuAGuAAUGGuAUCUTsT  589 AD-14088  22%  8% GAuuGuucAucAAuuGGcGTsT  590 CGCcAAUUGAUGAAcAAUCTsT  591 AD-14089  70% 12% GcuuucuccucGGcucAcuTsT  592 AGuGAGCCGAGGAGAAAGCTsT  593 AD-14090  79% 11% GGAGGAuuGGcuGAcAAGATsT  594 UCUUGUcAGCcAAUCCUCCTsT  595 AD-14091  29%  3% uAAuGAAGAGuAuAccuGGTsT  596 CcAGGuAuACUCUUcAUuATsT  597 AD-14092  23%  2% uuucAccAAAccAuuuGuATsT  598 uAcAAAUGGUUUGGUGAAATsT  599 AD-14093  60%  2% cuuAuuAAGGAGuAuAcGGTsT  600 CCGuAuACUCCUuAAuAAGTsT  601 AD-14094  11%  3% GAAAucAGAuGGAcGuAAGTsT  602 CUuACGUCcAUCUGAUUUCTsT  603 AD-14095  10%  2% cAGAuGucAGcAuAAGcGATsT  604 UCGCUuAUGCUGAcAUCUGTsT  605 AD-14096  27%  2% AucuAAcccuAGuuGuAucTsT  606 GAuAcAACuAGGGUuAGAUTsT  607 AD-14097  45%  6% AAGAGcuuGuuAAAAucGGTsT  608 CCGAUUUuAAcAAGCUCUUTsT  609 AD-14098  50% 10% uuAAGGAGuAuAcGGAGGATsT  610 UCCUCCGuAuACUCCUuAATsT  611 AD-14099  12%  4% uuGcAAuGuAAAuAcGuAuTsT  612 AuACGuAUUuAcAUUGcAATsT  613 AD-14100  49%  7% ucuAAcccuAGuuGuAuccTsT  614 GGAuAcAACuAGGGUuAGATsT  615 AD-14101  36%  1% cAuGuAucuuuuucucGAuTsT  616 AUCGAGAAAAAGAuAcAUGTsT  617 AD-14102  49%  3% GAuGucAGcAuAAGcGAuGTsT  618 cAUCGCUuAUGCUGAcAUCTsT  619 AD-14103  74%  5% ucccAAcAGGuAcGAcAccTsT  620 GGUGUCGuACCUGUUGGGATsT  621 AD-14104  27%  3% uGcucAcGAuGAGuuuAGuTsT  622 ACuAAACUcAUCGUGAGcATsT  623 AD-14105  34%  4% AGAGcuuGuuAAAAucGGATsT  624 UCCGAUUUuAAcAAGCUCUTsT  625 AD-14106   9%  2% GcGuAcAAGAAcAucuAuATsT  626 uAuAGAUGUUCUUGuACGCTsT  627 AD-14107   5%  1% GAGGuuGuAAGccAAuGuuTsT  628 AAcAUUGGCUuAcAACCUCTsT  629 AD-14108  15%  1% AAcAGGuAcGAcAccAcAGTsT  630 CUGUGGUGUCGuACCUGUUTsT  631 AD-14109  91%  2% AAcccuAGuuGuAucccucTsT  632 GAGGGAuAcAACuAGGGUUTsT  633 AD-14110  66%  5% GcAuAAGcGAuGGAuAAuATsT  634 uAUuAUCcAUCGCUuAUGCTsT  635 AD-14111  33%  3% AAGcGAuGGAuAAuAccuATsT  636 uAGGuAUuAUCcAUCGCUUTsT  637 AD-14112  51%  3% uGAuccuGuAcGAAAAGAATsT  638 UUCUUUUCGuAcAGGAUcATsT  639 AD-14113  22%  3% AAAAcAuuGGccGuucuGGTsT  640 CcAGAACGGCcAAUGUUUUTsT  641 AD-14114 117%  8% cuuGGAGGGcGuAcAAGAATsT  642 UUCUUGuACGCCCUCcAAGTsT  643 AD-14115  50%  8% GGcGuAcAAGAAcAucuAuTsT  644 AuAGAUGUUCUUGuACGCCTsT  645 AD-14116  14%  3% AcucuGAGuAcAuuGGAAuTsT  646 AUUCcAAUGuACUcAGAGUTsT  647 AD-14117  12%  4% uuAuuAAGGAGuAuAcGGATsT  648 UCCGuAuACUCCUuAAuAATsT  649 AD-14118  26%  4% uAAGGAGuAuAcGGAGGAGTsT  650 CUCCUCCGuAuACUCCUuATsT  651 AD-14119  24%  5% AAAucAAuAGucAAcuAAATsT  652 UUuAGUUGACuAUUGAUUUTsT  653 AD-14120   8%  1% AAucAAuAGucAAcuAAAGTsT  654 CUUuAGUUGACuAUUGAUUTsT  655 AD-14121  24%  2% uucucAGuAuAcuGuGuAATsT  656 UuAcAcAGuAuACUGAGAATsT  657 AD-14122  10%  1% uGuGAAAcAcucuGAuAAATsT  658 UUuAUcAGAGUGUUUcAcATsT  659 AD-14123   8%  1% AGAuGuGAAucucuGAAcATsT  660 UGUUcAGAGAUUcAcAUCUTsT  661 AD-14124   9%  2% AGGuuGuAAGccAAuGuuGTsT  662 cAAcAUUGGCUuAcAACCUTsT  663 AD-14125 114%  6% uGAGAAAucAGAuGGAcGuTsT  664 ACGUCcAUCUGAUUUCUcATsT  665 AD-14126   9%  1% AGAAAucAGAuGGAcGuAATsT  666 UuACGUCcAUCUGAUUUCUTsT  667 AD-14127  57%  6% AuAucccAAcAGGuAcGAcTsT  668 GUCGuACCUGUUGGGAuAUTsT  669 AD-14128 104%  6% cccAAcAGGuAcGAcAccATsT  670 UGGUGUCGuACCUGUUGGGTsT  671 AD-14129  21%  2% AGuAuAcuGAAGAAccucuTsT  672 AGAGGUUCUUcAGuAuACUTsT  673 AD-14130  57%  6% AuAuAuAucAGccGGGcGcTsT  674 GCGCCCGGCUGAuAuAuAUTsT  675 AD-14131  93%  6% AAucuAAcccuAGuuGuAuTsT  676 AuAcAACuAGGGUuAGAUUTsT  677 AD-14132  75%  8% cuAAcccuAGuuGuAucccTsT  678 GGGAuAcAACuAGGGUuAGTsT  679 AD-14133  66%  4% cuAGuuGuAucccuccuuuTsT  680 AAAGGAGGGAuAcAACuAGTsT  681 AD-14134  44%  6% AGAcAucuGAcuAAuGGcuTsT  682 AGCcAUuAGUcAGAUGUCUTsT  683 AD-14135  55%  6% GAAGcucAcAAuGAuuuAATsT  684 UuAAAUcAUUGUGAGCUUCTsT  685 AD-14136  29%  3% AcAuGuAucuuuuucucGATsT  686 UCGAGAAAAAGAuAcAUGUTsT  687 AD-14137  40%  3% ucGAuucAAAucuuAAcccTsT  688 GGGUuAAGAUUUGAAUCGATsT  689 AD-14138  39%  5% ucuuAAcccuuAGGAcucuTsT  690 AGAGUCCuAAGGGUuAAGATsT  691 AD-14139  71% 11% GcucAcGAuGAGuuuAGuGTsT  692 cACuAAACUcAUCGUGAGCTsT  693 AD-14140  43% 15% cAuAAGcGAuGGAuAAuAcTsT  694 GuAUuAUCcAUCGCUuAUGTsT  695 AD-14141  33%  6% AuAAGcGAuGGAuAAuAccTsT  696 GGuAUuAUCcAUCGCUuAUTsT  697 AD-14142  51% 14% ccuAAuAAAcuGcccucAGTsT  698 CUGAGGGcAGUUuAUuAGGTsT  699 AD-14143  42%  1% ucGGAAAGuuGAAcuuGGuTsT  700 ACcAAGUUcAACUUUCCGATsT  701 AD-14144   4%  4% GAAAAcAuuGGccGuucuGTsT  702 cAGAACGGCcAAUGUUUUCTsT  703 AD-14145  92%  5% AAGAcuGAucuucuAAGuuTsT  704 AACUuAGAAGAUcAGUCUUTsT  705 AD-14146  13%  2% GAGcuuGuuAAAAucGGAATsT  706 UUCCGAUUUuAAcAAGCUCTsT  707 AD-14147   8%  1% AcAuuGGccGuucuGGAGcTsT  708 GCUCcAGAACGGCcAAUGUTsT  709 AD-14148  80%  7% AAGAAcAucuAuAAuuGcATsT  710 UGcAAUuAuAGAUGUUCUUTsT  711 AD-14149  44%  7% AAAuGuGucuAcucAuGuuTsT  712 AAcAUGAGuAGAcAcAUUUTsT  713 AD-14150  32% 29% uGucuAcucAuGuuucucATsT  714 UGAGAAAcAUGAGuAGAcATsT  715 AD-14151  75% 11% GuAuAcuGuGuAAcAAucuTsT  716 AGAUUGUuAcAcAGuAuACTsT  717 AD-14152   8%  5% uAuAcuGuGuAAcAAucuATsT  718 uAGAUUGUuAcAcAGuAuATsT  719 AD-14153  17% 11% cuuAGuAGuGuccAGGAAATsT  720 UUUCCUGGAcACuACuAAGTsT  721 AD-14154  16%  4% ucAGAuGGAcGuAAGGcAGTsT  722 CUGCCUuACGUCcAUCUGATsT  723 AD-14155  11%  1% AGAuAAAuuGAuAGcAcAATsT  724 UUGUGCuAUcAAUUuAUCUTsT  725 AD-14156  10%  1% cAAcAGGuAcGAcAccAcATsT  726 UGUGGUGUCGuACCUGUUGTsT  727 AD-14157  29%  3% uGcAAuGuAAAuAcGuAuuTsT  728 AAuACGuAUUuAcAUUGcATsT  729 AD-14158  51%  3% AGucAGAAuuuuAucuAGATsT  730 UCuAGAuAAAAUUCUGACUTsT  731 AD-14159  53%  5% cuAGAAAucuuuuAAcAccTsT  732 GGUGUuAAAAGAUUUCuAGTsT  733 AD-14160  40%  3% AAuAAAucuAAcccuAGuuTsT  734 AACuAGGGUuAGAUUuAUUTsT  735 AD-14161  83%  7% AAuuuucuGcucAcGAuGATsT  736 UcAUCGUGAGcAGAAAAUUTsT  737 AD-14162  44%  6% GcccucAGuAAAuccAuGGTsT  738 CcAUGGAUUuACUGAGGGCTsT  739 AD-14163  57%  3% AcGuuuAAAAcGAGAucuuTsT  740 AAGAUCUCGUUUuAAACGUTsT  741 AD-14164   4%  1% AGGAGAuAGAAcGuuuAAATsT  742 UUuAAACGUUCuAUCUCCUTsT  743 AD-14165  11%  1% GAccGucAuGGcGucGcAGTsT  744 CUGCGACGCcAUGACGGUCTsT  745 AD-14166  90%  5% AccGucAuGGcGucGcAGcTsT  746 GCUGCGACGCcAUGACGGUTsT  747 AD-14167  49%  1% GAAcGuuuAAAAcGAGAucTsT  748 GAUCUCGUUUuAAACGUUCTsT  749 AD-14168  12%  2% uuGAGcuuAAcAuAGGuAATsT  750 UuACCuAUGUuAAGCUcAATsT  751 AD-14169  66%  4% AcuAAAuuGAucucGuAGATsT  752 UCuACGAGAUcAAUUuAGUTsT  753 AD-14170  52%  6% ucGuAGAAuuAucuuAAuATsT  754 uAUuAAGAuAAUUCuACGATsT  755 AD-14171  42%  4% GGAGAuAGAAcGuuuAAAATsT  756 UUUuAAACGUUCuAUCUCCTsT  757 AD-14172   3%  1% AcAAcuuAuuGGAGGuuGuTsT  758 AcAACCUCcAAuAAGUUGUTsT  759 AD-14173  29%  2% uGAGcuuAAcAuAGGuAAATsT  760 UUuACCuAUGUuAAGCUcATsT  761 AD-14174  69%  2% AucucGuAGAAuuAucuuATsT  762 uAAGAuAAUUCuACGAGAUTsT  763 AD-14175  53%  3% cuGcGuGcAGucGGuccucTsT  764 GAGGACCGACUGcACGcAGTsT  765 AD-14176 111%  4% cAcGcAGcGcccGAGAGuATsT  766 uACUCUCGGGCGCUGCGUGTsT  767 AD-14177  87%  6% AGuAccAGGGAGAcuccGGTsT  768 CCGGAGUCUCCCUGGuACUTsT  769 AD-14178  59%  2% AcGGAGGAGAuAGAAcGuuTsT  770 AACGUUCuAUCUCCUCCGUTsT  771 AD-14179   9%  2% AGAAcGuuuAAAAcGAGAuTsT  772 AUCUCGUUUuAAACGUUCUTsT  773 AD-14180  43%  2% AAcGuuuAAAAcGAGAucuTsT  774 AGAUCUCGUUUuAAACGUUTsT  775 AD-14181  70% 10% AGcuuGAGcuuAAcAuAGGTsT  776 CCuAUGUuAAGCUcAAGCUTsT  777 AD-14182 100%  7% AGcuuAAcAuAGGuAAAuATsT  778 uAUUuACCuAUGUuAAGCUTsT  779 AD-14183  60%  5% uAGAGcuAcAAAAccuAucTsT  780 GAuAGGUUUUGuAGCUCuATsT  781 AD-14184 129%  6% uAGuuGuAucccuccuuuATsT  782 uAAAGGAGGGAuAcAACuATsT  783 AD-14185  62%  4% AccAcccAGAcAucuGAcuTsT  784 AGUcAGAUGUCUGGGUGGUTsT  785 AD-14186  42%  3% AGAAAcuAAAuuGAucucGTsT  786 CGAGAUcAAUUuAGUUUCUTsT  787 AD-14187 123% 12% ucucGuAGAAuuAucuuAATsT  788 UuAAGAuAAUUCuACGAGATsT  789 AD-14188  38%  2% cAAcuuAuuGGAGGuuGuATsT  790 uAcAACCUCcAAuAAGUUGTsT  791 AD-14189  13%  1% uuGuAucccuccuuuAAGuTsT  792 ACUuAAAGGAGGGAuAcAATsT  793 AD-14190  59%  3% ucAcAAcuuAuuGGAGGuuTsT  794 AACCUCcAAuAAGUUGUGATsT  795 AD-14191  93%  3% AGAAcuGuAcucuucucAGTsT  796 CUGAGAAGAGuAcAGUUCUTsT  797 AD-14192  45%  5% GAGcuuAAcAuAGGuAAAuTsT  798 AUUuACCuAUGUuAAGCUCTsT  799 AD-14193  57%  3% cAccAAcAucuGuccuuAGTsT  800 CuAAGGAcAGAUGUUGGUGTsT  801 AD-14194  51%  4% AAAGcccAcuuuAGAGuAuTsT  802 AuACUCuAAAGUGGGCUUUTsT  803 AD-14195  77%  5% AAGcccAcuuuAGAGuAuATsT  804 uAuACUCuAAAGUGGGCUUTsT  805 AD-14196  42%  6% GAccuuAuuuGGuAAucuGTsT  806 cAGAUuACcAAAuAAGGUCTsT  807 AD-14197  15%  2% GAuuAAuGuAcucAAGAcuTsT  808 AGUCUUGAGuAcAUuAAUCTsT  809 AD-14198  12%  2% cuuuAAGAGGccuAAcucATsT  810 UGAGUuAGGCCUCUuAAAGTsT  811 AD-14199  18%  2% uuAAAccAAAcccuAuuGATsT  812 UcAAuAGGGUUUGGUUuAATsT  813 AD-14200  72%  9% ucuGuuGGAGAucuAuAAuTsT  814 AUuAuAGAUCUCcAAcAGATsT  815 AD-14201   9%  3% cuGAuGuuucuGAGAGAcuTsT  816 AGUCUCUcAGAAAcAUcAGTsT  817 AD-14202  25%  3% GcAuAcucuAGucGuucccTsT  818 GGGAACGACuAGAGuAUGCTsT  819 AD-14203  21%  1% GuuccuuAucGAGAAucuATsT  820 uAGAUUCUCGAuAAGGAACTsT  821 AD-14204   4%  2% GcAcuuGGAucucucAcAuTsT  822 AUGUGAGAGAUCcAAGUGCTsT  823 AD-14205   5%  1% AAAAAAGGAAcuAGAuGGcTsT  824 GCcAUCuAGUUCCUUUUUUTsT  825 AD-14206  79%  6% AGAGcAGAuuAccucuGcGTsT  826 CGcAGAGGuAAUCUGCUCUTsT  827 AD-14207  55%  2% AGcAGAuuAccucuGcGAGTsT  828 CUCGcAGAGGuAAUCUGCUTsT  829 AD-14208 100%  4% cccuGAcAGAGuucAcAAATsT  830 UUUGUGAACUCUGUcAGGGTsT  831 AD-14209  34%  3% GuuuAccGAAGuGuuGuuuTsT  832 AAAcAAcACUUCGGuAAACTsT  833 AD-14210  13%  2% uuAcAGuAcAcAAcAAGGATsT  834 UCCUUGUUGUGuACUGuAATsT  835 AD-14211   9%  1% AcuGGAucGuAAGAAGGcATsT  836 UGCCUUCUuACGAUCcAGUTsT  837 AD-14212  20%  3% GAGcAGAuuAccucuGcGATsT  838 UCGcAGAGGuAAUCUGCUCTsT  839 AD-14213  48%  5% AAAAGAAGuuAGuGuAcGATsT  840 UCGuAcACuAACUUCUUUUTsT  841 AD-14214  28% 18% GAccAuuuAAuuuGGcAGATsT  842 UCUGCcAAAUuAAAUGGUCTsT  843 AD-14215 132%  0% GAGAGGAGuGAuAAuuAAATsT  844 UUuAAUuAUcACUCCUCUCTsT  845 AD-14216   3%  0% cuGGAGGAuuGGcuGAcAATsT  846 UUGUcAGCcAAUCCUCcAGTsT  847 AD-14217  19%  1% cucuAGucGuucccAcucATsT  848 UGAGUGGGAACGACuAGAGTsT  849 AD-14218  67%  8% GAuAccAuuAcuAcAGuAGTsT  850 CuACUGuAGuAAUGGuAUCTsT  851 AD-14219  76%  4% uucGucuGcGAAGAAGAAATsT  852 UUUCUUCUUCGcAGACGAATsT  853 AD-14220  33%  8% GAAAAGAAGuuAGuGuAcGTsT  854 CGuAcACuAACUUCUUUUCTsT  855 AD-14221  25%  2% uGAuGuuuAccGAAGuGuuTsT  856 AAcACUUCGGuAAAcAUcATsT  857 AD-14222   7%  2% uGuuuGuccAAuucuGGAuTsT  858 AUCcAGAAUUGGAcAAAcATsT  859 AD-14223  19%  2% AuGAAGAGuAuAccuGGGATsT  860 UCCcAGGuAuACUCUUcAUTsT  861 AD-14224  13%  1% GcuAcucuGAuGAAuGcAuTsT  862 AUGcAUUcAUcAGAGuAGCTsT  863 AD-14225  15%  2% GcccuuGuAGAAAGAAcAcTsT  864 GUGUUCUUUCuAcAAGGGCTsT  865 AD-14226  11%  0% ucAuGuuccuuAucGAGAATsT  866 UUCUCGAuAAGGAAcAUGATsT  867 AD-14227   5%  1% GAAuAGGGuuAcAGAGuuGTsT  868 cAACUCUGuAACCCuAUUCTsT  869 AD-14228  34%  3% cAAAcuGGAucGuAAGAAGTsT  870 CUUCUuACGAUCcAGUUUGTsT  871 AD-14229  15%  2% cuuAuuuGGuAAucuGcuGTsT  872 cAGcAGAUuACcAAAuAAGTsT  873 AD-14230  20%  1% AGcAAuGuGGAAAccuAAcTsT  874 GUuAGGUUUCcAcAUUGCUTsT  875 AD-14231  18%  1% AcAAuAAAGcAGAcccAuuTsT  876 AAUGGGUCUGCUUuAUUGUTsT  877 AD-14232  21%  1% AAccAcuuAGuAGuGuccATsT  878 UGGAcACuACuAAGUGGUUTsT  879 AD-14233 106% 12% AGucAAGAGccAucuGuAGTsT  880 CuAcAGAUGGCUCUUGACUTsT  881 AD-14234  35%  3% cucccuAGAcuucccuAuuTsT  882 AAuAGGGAAGUCuAGGGAGTsT  883 AD-14235  48%  4% AuAGcuAAAuuAAAccAAATsT  884 UUUGGUUuAAUUuAGCuAUTsT  885 AD-14236  23%  3% uGGcuGGuAuAAuuccAcGTsT  886 CGUGGAAUuAuACcAGCcATsT  887 AD-14237  79%  9% uuAuuuGGuAAucuGcuGuTsT  888 AcAGcAGAUuACcAAAuAATsT  889 AD-14238  92%  7% AAcuAGAuGGcuuucucAGTsT  890 CUGAGAAAGCcAUCuAGUUTsT  891 AD-14239  20%  2% ucAuGGcGucGcAGccAAATsT  892 UUUGGCUGCGACGCcAUGATsT  893 AD-14240  71%  6% AcuGGAGGAuuGGcuGAcATsT  894 UGUcAGCcAAUCCUCcAGUTsT  895 AD-14241  14%  1% cuAuAAuuGcAcuAucuuuTsT  896 AAAGAuAGUGcAAUuAuAGTsT  897 AD-14242  11%  2% AAAGGucAccuAAuGAAGATsT  898 UCUUcAUuAGGUGACCUUUTsT  899 AD-14243  11%  1% AuGAAuGcAuAcucuAGucTsT  900 GACuAGAGuAUGcAUUcAUTsT  901 AD-14244  15%  2% AAcAuAuuGAAuAAGccuGTsT  902 cAGGCUuAUUcAAuAUGUUTsT  903 AD-14245  80%  7% AAGAAGGcAGuuGAccAAcTsT  904 GUUGGUcAACUGCCUUCUUTsT  905 AD-14246  57%  5% GAuAcuAAAAGAAcAAucATsT  906 UGAUUGUUCUUUuAGuAUCTsT  907 AD-14247   9%  3% AuAcuGAAAAucAAuAGucTsT  908 GACuAUUGAUUUUcAGuAUTsT  909 AD-14248  39%  4% AAAAAGGAAcuAGAuGGcuTsT  910 AGCcAUCuAGUUCCUUUUUTsT  911 AD-14249  64%  2% GAAcuAGAuGGcuuucucATsT  912 UGAGAAAGCcAUCuAGUUCTsT  913 AD-14250  18%  2% GAAAccuAAcuGAAGAccuTsT  914 AGGUCUUcAGUuAGGUUUCTsT  915 AD-14251  56%  6% uAcccAucAAcAcuGGuAATsT  916 UuACcAGUGUUGAUGGGuATsT  917 AD-14252  48%  6% AuuuuGAuAucuAcccAuuTsT  918 AAUGGGuAGAuAUcAAAAUTsT  919 AD-14253  39%  5% AucccuAuAGuucAcuuuGTsT  920 cAAAGUGAACuAuAGGGAUTsT  921 AD-14254  44%  8% AuGGGcuAuAAuuGcAcuATsT  922 uAGUGcAAUuAuAGCCcAUTsT  923 AD-14255 108%  8% AGAuuAccucuGcGAGcccTsT  924 GGGCUCGcAGAGGuAAUCUTsT  925 AD-14256 108%  6% uAAuuccAcGuAcccuucATsT  926 UGAAGGGuACGUGGAAUuATsT  927 AD-14257  23%  2% GucGuucccAcucAGuuuuTsT  928 AAAACuGAGuGGGAACGACTsT  929 AD-14258  21%  3% AAAucAAucccuGuuGAcuTsT  930 AGUcAAcAGGGAUUGAUUUTsT  931 AD-14259  19%  2% ucAuAGAGcAAAGAAcAuATsT  932 uAUGUUCUUUGCUCuAUGATsT  933 AD-14260  10%  1% uuAcuAcAGuAGcAcuuGGTsT  934 CcAAGUGCuACUGuAGuAATsT  935 AD-14261  76%  3% AuGuGGAAAccuAAcuGAATsT  936 UUcAGUuAGGUUUCcAcAUTsT  937 AD-14262  13%  2% uGuGGAAAccuAAcuGAAGTsT  938 CUUcAGUuAGGUUUCcAcATsT  939 AD-14263  14%  2% ucuuccuuAAAuGAAAGGGTsT  940 CCCUUUcAUUuAAGGAAGATsT  941 AD-14264  65%  3% uGAAGAAccucuAAGucAATsT  942 UUGACUuAGAGGUUCUUcATsT  943 AD-14265  13%  1% AGAGGucuAAAGuGGAAGATsT  944 UCUUCcACUUuAGACCUCUTsT  945 AD-14266  18%  3% AuAucuAcccAuuuuucuGTsT  946 cAGAAAAAUGGGuAGAuAUTsT  947 AD-14267  50%  9% uAAGccuGAAGuGAAucAGTsT  948 CUGAUUcACUUcAGGCUuATsT  949 AD-14268  13%  3% AGAuGcAGAccAuuuAAuuTsT  950 AAUuAAAUGGUCUGcAUCUTsT  951 AD-14269  19%  4% AGuGuuGuuuGuccAAuucTsT  952 GAAUUGGAcAAAcAAcACUTsT  953 AD-14270  11%  2% cuAuAAuGAAGAGcuuuuuTsT  954 AAAAAGCUCUUcAUuAuAGTsT  955 AD-14271  11%  1% AGAGGAGuGAuAAuuAAAGTsT  956 CUUuAAUuAUcACUCCUCUTsT  957 AD-14272   7%  1% uuucucuGuuAcAAuAcAuTsT  958 AUGuAUUGuAAcAGAGAAATsT  959 AD-14273  14%  2% AAcAucuAuAAuuGcAAcATsT  960 UGUUGcAAUuAuAGAUGUUTsT  961 AD-14274  73%  4% uGcuAGAAGuAcAuAAGAcTsT  962 GUCUuAUGuACUUCuAGcATsT  963 AD-14275  10%  1% AAuGuAcucAAGAcuGAucTsT  964 GAUcAGUCUUGAGuAcAUUTsT  965 AD-14276  89%  2% GuAcucAAGAcuGAucuucTsT  966 GAAGAUcAGUCUUGAGuACTsT  967 AD-14277   7%  1% cAcucuGAuAAAcucAAuGTsT  968 cAUUGAGUUuAUcAGAGUGTsT  969 AD-14278  12%  1% AAGAGcAGAuuAccucuGcTsT  970 GcAGAGGuAAUCUGCUCUUTsT  971 AD-14279 104%  3% ucuGcGAGcccAGAucAAcTsT  972 GUUGAUCUGGGCUCGcAGATsT  973 AD-14280  21%  2% AAcuuGAGccuuGuGuAuATsT  974 uAuAcAcAAGGCUcAAGUUTsT  975 AD-14281  43%  3% GAAuAuAuAuAucAGccGGTsT  976 CCGGCUGAuAuAuAuAUUCTsT  977 AD-14282  45%  6% uGucAucccuAuAGuucAcTsT  978 GUGAACuAuAGGGAUGAcATsT  979 AD-14283  35%  5% GAucuGGcAAccAuAuuucTsT  980 GAAAuAUGGUUGCcAGAUCTsT  981 AD-14284  58%  3% uGGcAAccAuAuuucuGGATsT  982 UCcAGAAAuAUGGUUGCcATsT  983 AD-14285  48%  3% GAuGuuuAccGAAGuGuuGTsT  984 cAAcACUUCGGuAAAcAUCTsT  985 AD-14286  49%  3% uuccuuAucGAGAAucuAATsT  986 UuAGAUUCUCGAuAAGGAATsT  987 AD-14287   6%  1% AGcuuAAuuGcuuucuGGATsT  988 UCcAGAAAGcAAUuAAGCUTsT  989 AD-14288  50%  2% uuGcuAuuAuGGGAGAccATsT  990 UGGUCUCCcAuAAuAGcAATsT  991 AD-14289  48%  1% GucAuGGcGucGcAGccAATsT  992 UUGGCUGCGACGCcAUGACTsT  993 AD-14290 112%  7% uAAuuGcAcuAucuuuGcGTsT  994 CGcAAAGAuAGUGcAAUuATsT  995 AD-14291  77%  2% cuAucuuuGcGuAuGGccATsT  996 UGGCcAuACGcAAAGAuAGTsT  997 AD-14292  80%  6% ucccuAuAGuucAcuuuGuTsT  998 AcAAAGUGAACuAuAGGGATsT  999 AD-14293  58%  2% ucAAccuuuAAuucAcuuGTsT 1000 cAAGUGAAUuAAAGGUUGATsT 1001 AD-14294  77%  2% GGcAAccAuAuuucuGGAATsT 1002 UUCcAGAAAuAUGGUUGCCTsT 1003 AD-14295  62%  2% AuGuAcucAAGAcuGAucuTsT 1004 AGAUcAGUCUUGAGuAcAUTsT 1005 AD-14296  59%  4% GcAGAccAuuuAAuuuGGcTsT 1006 GCcAAAUuAAAUGGUCUGCTsT 1007 AD-14297  37%  1% ucuGAGAGAcuAcAGAuGuTsT 1008 AcAUCUGuAGUCUCUcAGATsT 1009 AD-14298  21%  1% uGcucAuAGAGcAAAGAAcTsT 1010 GUUCUUUGCUCuAUGAGcATsT 1011 AD-14299   6%  1% AcAuAAGAccuuAuuuGGuTsT 1012 ACcAAAuAAGGUCUuAUGUTsT 1013 AD-14300  17%  2% uuuGuGcuGAuucuGAuGGTsT 1014 CcAUcAGAAUcAGcAcAAATsT 1015 AD-14301  97%  6% ccAucAAcAcuGGuAAGAATsT 1016 UUCUuACcAGUGUUGAUGGTsT 1017 AD-14302  13%  1% AGAcAAuuccGGAuGuGGATsT 1018 UCcAcAUCCGGAAUUGUCUTsT 1019 AD-14303  13%  3% GAAcuuGAGccuuGuGuAuTsT 1020 AuAcAcAAGGCUcAAGUUCTsT 1021 AD-14304  38%  2% uAAuuuGGcAGAGcGGAAATsT 1022 UUUCCGCUCUGCcAAAUuATsT 1023 AD-14305  14%  2% uGGAuGAAGuuAuuAuGGGTsT 1024 CCcAuAAuAACUUcAUCcATsT 1025 AD-14306  22%  4% AucuAcAuGAAcuAcAAGATsT 1026 UCUUGuAGUUcAUGuAGAUTsT 1027 AD-14307  26%  6% GGuAuuuuuGAucuGGcAATsT 1028 UUGCcAGAUcAAAAAuACCTsT 1029 AD-14308  62%  8% cuAAuGAAGAGuAuAccuGTsT 1030 cAGGuAuACUCUUcAUuAGTsT 1031 AD-14309  52%  5% uuuGAGAAAcuuAcuGAuATsT 1032 uAUcAGuAAGUUUCUcAAATsT 1033 AD-14310  32%  3% cGAuAAGAuAGAAGAucAATsT 1034 UUGAUCUUCuAUCUuAUCGTsT 1035 AD-14311  23%  2% cuGGcAAccAuAuuucuGGTsT 1036 CcAGAAAuAUGGUUGCcAGTsT 1037 AD-14312  49%  6% uAGAuAccAuuAcuAcAGuTsT 1038 ACUGuAGuAAUGGuAUCuATsT 1039 AD-14313  69%  4% GuAuuAAAuuGGGuuucAuTsT 1040 AUGAAACCcAAUUuAAuACTsT 1041 AD-14314  52%  3% AAGAccuuAuuuGGuAAucTsT 1042 GAUuACcAAAuAAGGUCUUTsT 1043 AD-14315  66%  4% GcuGuuGAuAAGAGAGcucTsT 1044 GAGCUCUCUuAUcAAcAGCTsT 1045 AD-14316  19%  4% uAcucAuGuuucucAGAuuTsT 1046 AAUCUGAGAAAcAUGAGuATsT 1047 AD-14317  16%  5% cAGAuGGAcGuAAGGcAGcTsT 1048 GCUGCCUuACGUCcAUCUGTsT 1049 AD-14318  52% 11% uAucccAAcAGGuAcGAcATsT 1050 UGUCGuACCUGUUGGGAuATsT 1051 AD-14319  28% 11% cAuuGcuAuuAuGGGAGAcTsT 1052 GUCUCCcAuAAuAGcAAUGTsT 1053 AD-14320  52% 10% cccucAGuAAAuccAuGGuTsT 1054 ACcAUGGAUUuACUGAGGGTsT 1055 AD-14321  53%  6% GGucAuuAcuGcccuuGuATsT 1056 uAcAAGGGcAGuAAUGACCTsT 1057 AD-14322  20%  2% AAccAcucAAAAAcAuuuGTsT 1058 cAAAUGUUUUUGAGUGGUUTsT 1059 AD-14323 116%  6% uuuGcAAGuuAAuGAAucuTsT 1060 AGAUUcAUuAACUUGcAAATsT 1061 AD-14324  14%  2% uuAuuuucAGuAGucAGAATsT 1062 UUCUGACuACUGAAAAuAATsT 1063 AD-14325  50%  2% uuuucucGAuucAAAucuuTsT 1064 AAGAUUuGAAUCGAGAAAATsT 1065 AD-14326  47%  3% GuAcGAAAAGAAGuuAGuGTsT 1066 cACuAACUUCUUUUCGuACTsT 1067 AD-14327  18%  2% uuuAAAAcGAGAucuuGcuTsT 1068 AGcAAGAUCUCGUUUuAAATsT 1069 AD-14328  19%  1% GAAuuGAuuAAuGuAcucATsT 1070 UGAGuAcAUuAAUcAAUUCTsT 1071 AD-14329  94% 10% GAuGGAcGuAAGGcAGcucTsT 1072 GAGCUGCCUuACGUCcAUCTsT 1073 AD-14330  60%  4% cAucuGAcuAAuGGcucuGTsT 1074 cAGAGCcAUuAGUcAGAUGTsT 1075 AD-14331  54%  7% GuGAuccuGuAcGAAAAGATsT 1076 UCUUUUCGuAcAGGAUcACTsT 1077 AD-14332  22%  4% AGcucuuAuuAAGGAGuAuTsT 1078 AuACUCCUuAAuAAGAGCUTsT 1079 AD-14333  70% 10% GcucuuAuuAAGGAGuAuATsT 1080 uAuACUCCUuAAuAAGAGCTsT 1081 AD-14334  18%  3% ucuuAuuAAGGAGuAuAcGTsT 1082 CGuAuACUCCUuAAuAAGATsT 1083 AD-14335  38%  6% uAuuAAGGAGuAuAcGGAGTsT 1084 CUCCGuAuACUCCUuAAuATsT 1085 AD-14336  16%  3% cuGcAGcccGuGAGAAAAATsT 1086 UUUUUCUcACGGGCUGcAGTsT 1087 AD-14337  65%  4% ucAAGAcuGAucuucuAAGTsT 1088 CUuAGAAGAUcAGUCUUGATsT 1089 AD-14338  18%  0% cuucuAAGuucAcuGGAAATsT 1090 UUUCcAGUGAACUuAGAAGTsT 1091 AD-14339  20%  4% uGcAAGuuAAuGAAucuuuTsT 1092 AAAGAUUcAUuAACUUGcATsT 1093 AD-14340  24%  1% AAucuAAGGAuAuAGucAATsT 1094 UUGACuAuAUCCUuAGAUUTsT 1095 AD-14341  27%  3% AucucuGAAcAcAAGAAcATsT 1096 UGUUCUUGUGUUcAGAGAUTsT 1097 AD-14342  13%  1% uucuGAAcAGuGGGuAucuTsT 1098 AGAuACCcACUGUUcAGAATsT 1099 AD-14343  19%  1% AGuuAuuuAuAcccAucAATsT 1100 UUGAUGGGuAuAAAuAACUTsT 1101 AD-14344  23%  2% AuGcuAAAcuGuucAGAAATsT 1102 UUUCUGAAcAGUUuAGcAUTsT 1103 AD-14345  21%  4% cuAcAGAGcAcuuGGuuAcTsT 1104 GuAACcAAGUGCUCUGuAGTsT 1105 AD-14346  18%  2% uAuAuAucAGccGGGcGcGTsT 1106 CGCGCCCGGCUGAuAuAuATsT 1107 AD-14347  67%  2% AuGuAAAuAcGuAuuucuATsT 1108 uAGAAAuACGuAUUuAcAUTsT 1109 AD-14348  39%  3% uuuuucucGAuucAAAucuTsT 1110 AGAUUuGAAUCGAGAAAAATsT 1111 AD-14349  83%  6% AAucuuAAcccuuAGGAcuTsT 1112 AGUCCuAAGGGUuAAGAUUTsT 1113 AD-14350  54%  2% ccuuAGGAcucuGGuAuuuTsT 1114 AAAuACcAGAGUCCuAAGGTsT 1115 AD-14351  57%  8% AAuAAAcuGcccucAGuAATsT 1116 UuACUGAGGGcAGUUuAUUTsT 1117 AD-14352  82%  3% GAuccuGuAcGAAAAGAAGTsT 1118 CUUCUUUUCGuAcAGGAUCTsT 1119 AD-14353   2%  1% AAuGuGAuccuGuAcGAAATsT 1120 UUUCGuAcAGGAUcAcAUUTsT 1121 AD-14354  18% 11% GuGAAAAcAuuGGccGuucTsT 1122 GAACGGCcAAUGUUUUcACTsT 1123 AD-14355   2%  1% cuuGAGGAAAcucuGAGuATsT 1124 uACUcAGAGUUUCCUcAAGTsT 1125 AD-14356   8%  2% cGuuuAAAAcGAGAucuuGTsT 1126 cAAGAUCUCGUUUuAAACGTsT 1127 AD-14357   6%  3% uuAAAAcGAGAucuuGcuGTsT 1128 cAGcAAGAUCUCGUUUuAATsT 1129 AD-14358  98% 17% AAAGAuGuAucuGGucuccTsT 1130 GGAGACcAGAuAcAUCUUUTsT 1131 AD-14359  10%  1% cAGAAAAuGuGucuAcucATsT 1132 UGAGuAGAcAcAUUUUCUGTsT 1133 AD-14360   6%  4% cAGGAAuuGAuuAAuGuAcTsT 1134 GuAcAUuAAUcAAUUCCUGTsT 1135 AD-14361  30%  5% AGucAAcuAAAGcAuAuuuTsT 1136 AAAuAUGCUUuAGUUGACUTsT 1137 AD-14362  28%  2% uGuGuAAcAAucuAcAuGATsT 1138 UcAUGuAGAUUGUuAcAcATsT 1139 AD-14363  60%  6% AuAccAuuuGuuccuuGGuTsT 1140 ACcAAGGAAcAAAUGGuAUTsT 1141 AD-14364  12%  9% GcAGAAAucuAAGGAuAuATsT 1142 uAuAUCCUuAGAUUUCUGCTsT 1143 AD-14365   5%  2% uGGcuucucAcAGGAAcucTsT 1144 GAGUUCCUGUGAGAAGCcATsT 1145 AD-14366  28%  5% GAGAuGuGAAucucuGAAcTsT 1146 GUUcAGAGAUUcAcAUCUCTsT 1147 AD-14367  42%  4% uGuAAGccAAuGuuGuGAGTsT 1148 CUcAcAAcAUUGGCUuAcATsT 1149 AD-14368  93% 12% AGccAAuGuuGuGAGGcuuTsT 1150 AAGCCUcAcAAcAUUGGCUTsT 1151 AD-14369  65%  4% uuGuGAGGcuucAAGuucATsT 1152 UGAACUUGAAGCCUcAcAATsT 1153 AD-14370   5%  2% AGGcAGcucAuGAGAAAcATsT 1154 UGUUUCUcAUGAGCUGCCUTsT 1155 AD-14371  54%  5% AuAAAuuGAuAGcAcAAAATsT 1156 UUUUGUGCuAUcAAUUuAUTsT 1157 AD-14372   4%  1% AcAAAAucuAGAAcuuAAuTsT 1158 AUuAAGUUCuAGAUUUUGUTsT 1159 AD-14373   5%  1% GAuAucccAAcAGGuAcGATsT 1160 UCGuACCUGUUGGGAuAUCTsT 1161 AD-14374  92%  6% AAGuuAuuuAuAcccAucATsT 1162 UGAUGGGuAuAAAuAACUUTsT 1163 AD-14375  76%  4% uGuAAAuAcGuAuuucuAGTsT 1164 CuAGAAAuACGuAUUuAcATsT 1165 AD-14376  70%  5% ucuAGuuuucAuAuAAAGuTsT 1166 ACUUuAuAUGAAAACuAGATsT 1167 AD-14377  48%  4% AuAAAGuAGuucuuuuAuATsT 1168 uAuAAAAGAACuACUUuAUTsT 1169 AD-14378  48%  3% ccAuuuGuAGAGcuAcAAATsT 1170 UUUGuAGCUCuAcAAAUGGTsT 1171 AD-14379  44%  5% uAuuuucAGuAGucAGAAuTsT 1172 AUUCUGACuACUGAAAAuATsT 1173 AD-14380  35% 16% AAAucuAAcccuAGuuGuATsT 1174 uAcAACuAGGGUuAGAUUUTsT 1175 AD-14381  44%  5% cuuuAGAGuAuAcAuuGcuTsT 1176 AGcAAUGuAuACUCuAAAGTsT 1177 AD-14382  28%  1% AucuGAcuAAuGGcucuGuTsT 1178 AcAGAGCcAUuAGUcAGAUTsT 1179 AD-14383  55% 11% cAcAAuGAuuuAAGGAcuGTsT 1180 cAGUCCUuAAAUcAUUGUGTsT 1181 AD-14384  48%  9% ucuuuuucucGAuucAAAuTsT 1182 AUUuGAAUCGAGAAAAAGATsT 1183 AD-14385  36%  2% cuuuuucucGAuucAAAucTsT 1184 GAUUuGAAUCGAGAAAAAGTsT 1185 AD-14386  41%  7% AuuuucuGcucAcGAuGAGTsT 1186 CUcAUCGUGAGcAGAAAAUTsT 1187 AD-14387  38%  3% uuucuGcucAcGAuGAGuuTsT 1188 AACUcAUCGUGAGcAGAAATsT 1189 AD-14388  50%  4% AGAGcuAcAAAAccuAuccTsT 1190 GGAuAGGUUUUGuAGCUCUTsT 1191 AD-14389  98%  6% GAGccAAAGGuAcAccAcuTsT 1192 AGUGGUGuACCUUUGGCUCTsT 1193 AD-14390  43%  8% GccAAAGGuAcAccAcuAcTsT 1194 GuAGUGGUGuACCUUUGGCTsT 1195 AD-14391  48%  4% GAAcuGuAcucuucucAGcTsT 1196 GCUGAGAAGAGuAcAGUUCTsT 1197 AD-14392  44%  3% AGGuAAAuAucAccAAcAuTsT 1198 AUGUUGGUGAuAUUuACCUTsT 1199 AD-14393  37%  2% AGcuAcAAAAccuAuccuuTsT 1200 AAGGAuAGGUUUUGuAGCUTsT 1201 AD-14394 114%  7% uGuGAAAGcAuuuAAuuccTsT 1202 GGAAUuAAAUGCUUUcAcATsT 1203 AD-14395  55%  4% GcccAcuuuAGAGuAuAcATsT 1204 UGuAuACUCuAAAGUGGGCTsT 1205 AD-14396  49%  5% uGuGccAcAcuccAAGAccTsT 1206 GGUCUUGGAGUGUGGcAcATsT 1207 AD-14397  71%  6% AAAcuAAAuuGAucucGuATsT 1208 uACGAGAUcAAUUuAGUUUTsT 1209 AD-14398  81%  7% uGAucucGuAGAAuuAucuTsT 1210 AGAuAAUUCuACGAGAUcATsT 1211 AD-14399  38%  4% GcGuGcAGucGGuccuccATsT 1212 UGGAGGACCGACUGcACGCTsT 1213 AD-14400 106%  8% AAAGuuuAGAGAcAucuGATsT 1214 UcAGAUGUCUCuAAACUUUTsT 1215 AD-14401  47%  3% cAGAAGGAAuAuGuAcAAATsT 1216 UUUGuAcAuAUUCCUUCUGTsT 1217 AD-14402  31%  1% cGcccGAGAGuAccAGGGATsT 1218 UCCCUGGuACUCUCGGGCGTsT 1219 AD-14403 105%  4% cGGAGGAGAuAGAAcGuuuTsT 1220 AAACGUUCuAUCUCCUCCGTsT 1221 AD-14404   3%  1% AGAuAGAAcGuuuAAAAcGTsT 1222 CGUUUuAAACGUUCuAUCUTsT 1223 AD-14405  15%  1% GGAAcAGGAAcuucAcAAcTsT 1224 GUuGuGAAGUUCCuGUUCCTsT 1225 AD-14406  44%  5% GuGAGccAAAGGuAcAccATsT 1226 UGGUGuACCUUUGGCUcACTsT 1227 AD-14407  41%  4% AuccucccuAGAcuucccuTsT 1228 AGGGAAGUCuAGGGAGGAUTsT 1229 AD-14408 104%  3% cAcAcuccAAGAccuGuGcTsT 1230 GcAcAGGUCUUGGAGUGUGTsT 1231 AD-14409  67%  4% AcAGAAGGAAuAuGuAcAATsT 1232 UUGuAcAuAUUCCUUCUGUTsT 1233 AD-14410  22%  1% uuAGAGAcAucuGAcuuuGTsT 1234 cAAAGUcAGAUGUCUCuAATsT 1235 AD-14411  29%  3% AAuuGAucucGuAGAAuuATsT 1236 uAAUUCuACGAGAUcAAUUTsT 1237 AD-14412  31%  4%

dsRNA Targeting the VEGF Gene

Four hundred target sequences were identified within exons 1-5 of the VEGF-Al21 mRNA sequence. reference transcript is: NM_(—)003376.

(SEQ ID NO: 1539)   1 augaacuuuc ugcugucuug ggugcauugg agccuugccu ugcugcucua ccuccaccau  61 gccaaguggu cccaggcugc acccauggca gaaggaggag ggcagaauca ucacgaagug 121 gugaaguuca uggaugucua ucagcgcagc uacugccauc caaucgagac ccugguggac 181 aucuuccagg aguacccuga ugagaucgag uacaucuuca agccauccug ugugccccug 241 augcgaugcg ggggcugcug caaugacgag ggccuggagu gugugcccac ugaggagucc 301 aacaucacca ugcagauuau gcggaucaaa ccucaccaag gccagcacau aggagagaug 361 agcuuccuac agcacaacaa augugaaugc agaccaaaga aagauagagc aagacaagaa 421 aaaugugaca agccgaggcg guga

Table 4a includes the identified target sequences. Corresponding siRNAs targeting these sequences were subjected to a bioinformatics screen.

To ensure that the sequences were specific to VEGF sequence and not to sequences from any other genes, the target sequences were checked against the sequences in Genbank using the BLAST search engine provided by NCBI. The use of the BLAST algorithm is described in Altschul et al., J. Mol. Biol. 215:403, 1990; and Altschul and Gish, Meth. Enzymol. 266:460, 1996.

siRNAs were also prioritized for their ability to cross react with monkey, rat and human VEGF sequences.

Of these 400 potential target sequences 80 were selected for analysis by experimental screening in order to identify a small number of lead candidates. A total of 114 siRNA molecules were designed for these 80 target sequences 114 (Table 4b).

TABLE 4a Target sequences in VEGF-121 SEQ position TARGET SEQUENCE IN ID in VEGF- VEGF121 mRNA NO: 121 ORF 5′ to 3′ 1540   1 AUGAACUUUCUGCUGUCUUGGGU 1541   2 UGAACUUUCUGCUGUCUUGGGUG 1542   3 GAACUUUCUGCUGUCUUGGGUGC 1543   4 AACUUUCUGCUGUCUUGGGUGCA 1544   5 ACUUUCUGCUGUCUUGGGUGCAU 1545   6 CUUUCUGCUGUCUUGGGUGCAUU 1546   7 UUUCUGCUGUCUUGGGUGCAUUG 1547   8 UUCUGCUGUCUUGGGUGCAUUGG 1548   9 UCUGCUGUCUUGGGUGCAUUGGA 1549  10 CUGCUGUCUUGGGUGCAUUGGAG 1550  11 UGCUGUCUUGGGUGCAUUGGAGC 1551  12 GCUGUCUUGGGUGCAUUGGAGCC 1552  13 CUGUCUUGGGUGCAUUGGAGCCU 1553  14 UGUCUUGGGUGCAUUGGAGCCUU 1554  15 GUCUUGGGUGCAUUGGAGCCUUG 1555  16 UCUUGGGUGCAUUGGAGCCUUGC 1556  17 CUUGGGUGCAUUGGAGCCUUGCC 1557  18 UUGGGUGCAUUGGAGCCUUGCCU 1558  19 UGGGUGCAUUGGAGCCUUGCCUU 1559  20 GGGUGCAUUGGAGCCUUGCCUUG 1560  21 GGUGCAUUGGAGCCUUGCCUUGC 1561  22 GUGCAUUGGAGCCUUGCCUUGCU 1562  23 UGCAUUGGAGCCUUGCCUUGCUG 1563  24 GCAUUGGAGCCUUGCCUUGCUGC 1564  25 CAUUGGAGCCUUGCCUUGCUGCU 1565  26 AUUGGAGCCUUGCCUUGCUGCUC 1566  27 UUGGAGCCUUGCCUUGCUGCUCU 1567  28 UGGAGCCUUGCCUUGCUGCUCUA 1568  29 GGAGCCUUGCCUUGCUGCUCUAC 1569  30 GAGCCUUGCCUUGCUGCUCUACC 1570  31 AGCCUUGCCUUGCUGCUCUACCU 1571  32 GCCUUGCCUUGCUGCUCUACCUC 1572  33 CCUUGCCUUGCUGCUCUACCUCC 1573  34 CUUGCCUUGCUGCUCUACCUCCA 1574  35 UUGCCUUGCUGCUCUACCUCCAC 1575  36 UGCCUUGCUGCUCUACCUCCACC 1576  37 GCCUUGCUGCUCUACCUCCACCA 1577  38 CCUUGCUGCUCUACCUCCACCAU 1578  39 CUUGCUGCUCUACCUCCACCAUG 1579  40 UUGCUGCUCUACCUCCACCAUGC 1580  41 UGCUGCUCUACCUCCACCAUGCC 1581  42 GCUGCUCUACCUCCACCAUGCCA 1582  43 CUGCUCUACCUCCACCAUGCCAA 1583  44 UGCUCUACCUCCACCAUGCCAAG 1584  45 GCUCUACCUCCACCAUGCCAAGU 1585  46 CUCUACCUCCACCAUGCCAAGUG 1586  47 UCUACCUCCACCAUGCCAAGUGG 1587  48 CUACCUCCACCAUGCCAAGUGGU 1588  49 UACCUCCACCAUGCCAAGUGGUC 1589  50 ACCUCCACCAUGCCAAGUGGUCC 1590  51 CCUCCACCAUGCCAAGUGGUCCC 1591  52 CUCCACCAUGCCAAGUGGUCCCA 1592  53 UCCACCAUGCCAAGUGGUCCCAG 1593  54 CCACCAUGCCAAGUGGUCCCAGG 1594  55 CACCAUGCCAAGUGGUCCCAGGC 1595  56 ACCAUGCCAAGUGGUCCCAGGCU 1596  57 CCAUGCCAAGUGGUCCCAGGCUG 1597  58 CAUGCCAAGUGGUCCCAGGCUGC 1598  59 AUGCCAAGUGGUCCCAGGCUGCA 1599  60 UGCCAAGUGGUCCCAGGCUGCAC 1600  61 GCCAAGUGGUCCCAGGCUGCACC 1601  62 CCAAGUGGUCCCAGGCUGCACCC 1602  63 CAAGUGGUCCCAGGCUGCACCCA 1603  64 AAGUGGUCCCAGGCUGCACCCAU 1604  65 AGUGGUCCCAGGCUGCACCCAUG 1605  66 GUGGUCCCAGGCUGCACCCAUGG 1606  67 UGGUCCCAGGCUGCACCCAUGGC 1607  68 GGUCCCAGGCUGCACCCAUGGCA 1608  69 GUCCCAGGCUGCACCCAUGGCAG 1609  70 UCCCAGGCUGCACCCAUGGCAGA 1610  71 CCCAGGCUGCACCCAUGGCAGAA 1611  72 CCAGGCUGCACCCAUGGCAGAAG 1612  73 CAGGCUGCACCCAUGGCAGAAGG 1613  74 AGGCUGCACCCAUGGCAGAAGGA 1614  75 GGCUGCACCCAUGGCAGAAGGAG 1615  76 GCUGCACCCAUGGCAGAAGGAGG 1616  77 CUGCACCCAUGGCAGAAGGAGGA 1617  78 UGCACCCAUGGCAGAAGGAGGAG 1618  79 GCACCCAUGGCAGAAGGAGGAGG 1619  80 CACCCAUGGCAGAAGGAGGAGGG 1620  81 ACCCAUGGCAGAAGGAGGAGGGC 1621  82 CCCAUGGCAGAAGGAGGAGGGCA 1622  83 CCAUGGCAGAAGGAGGAGGGCAG 1623  84 CAUGGCAGAAGGAGGAGGGCAGA 1624  85 AUGGCAGAAGGAGGAGGGCAGAA 1625  86 UGGCAGAAGGAGGAGGGCAGAAU 1626  87 GGCAGAAGGAGGAGGGCAGAAUC 1627  88 GCAGAAGGAGGAGGGCAGAAUCA 1628  89 CAGAAGGAGGAGGGCAGAAUCAU 1629  90 AGAAGGAGGAGGGCAGAAUCAUC 1630  91 GAAGGAGGAGGGCAGAAUCAUCA 1631  92 AAGGAGGAGGGCAGAAUCAUCAC 1632  93 AGGAGGAGGGCAGAAUCAUCACG 1633  94 GGAGGAGGGCAGAAUCAUCACGA 1634  95 GAGGAGGGCAGAAUCAUCACGAA 1635  96 AGGAGGGCAGAAUCAUCACGAAG 1636  97 GGAGGGCAGAAUCAUCACGAAGU 1637  98 GAGGGCAGAAUCAUCACGAAGUG 1638  99 AGGGCAGAAUCAUCACGAAGUGG 1639 100 GGGCAGAAUCAUCACGAAGUGGU 1640 101 GGCAGAAUCAUCACGAAGUGGUG 1641 102 GCAGAAUCAUCACGAAGUGGUGA 1642 103 CAGAAUCAUCACGAAGUGGUGAA 1643 104 AGAAUCAUCACGAAGUGGUGAAG 1644 105 GAAUCAUCACGAAGUGGUGAAGU 1645 106 AAUCAUCACGAAGUGGUGAAGUU 1646 107 AUCAUCACGAAGUGGUGAAGUUC 1647 108 UCAUCACGAAGUGGUGAAGUUCA 1648 109 CAUCACGAAGUGGUGAAGUUCAU 1649 110 AUCACGAAGUGGUGAAGUUCAUG 1650 111 UCACGAAGUGGUGAAGUUCAUGG 1651 112 CACGAAGUGGUGAAGUUCAUGGA 1652 113 ACGAAGUGGUGAAGUUCAUGGAU 1653 114 CGAAGUGGUGAAGUUCAUGGAUG 1654 115 GAAGUGGUGAAGUUCAUGGAUGU 1655 116 AAGUGGUGAAGUUCAUGGAUGUC 1656 117 AGUGGUGAAGUUCAUGGAUGUCU 1657 118 GUGGUGAAGUUCAUGGAUGUCUA 1658 119 UGGUGAAGUUCAUGGAUGUCUAU 1659 120 GGUGAAGUUCAUGGAUGUCUAUC 1660 121 GUGAAGUUCAUGGAUGUCUAUCA 1661 122 UGAAGUUCAUGGAUGUCUAUCAG 1662 123 GAAGUUCAUGGAUGUCUAUCAGC 1663 124 AAGUUCAUGGAUGUCUAUCAGCG 1664 125 AGUUCAUGGAUGUCUAUCAGCGC 1665 126 GUUCAUGGAUGUCUAUCAGCGCA 1666 127 UUCAUGGAUGUCUAUCAGCGCAG 1667 128 UCAUGGAUGUCUAUCAGCGCAGC 1668 129 CAUGGAUGUCUAUCAGCGCAGCU 1669 130 AUGGAUGUCUAUCAGCGCAGCUA 1670 131 UGGAUGUCUAUCAGCGCAGCUAC 1671 132 GGAUGUCUAUCAGCGCAGCUACU 1672 133 GAUGUCUAUCAGCGCAGCUACUG 1673 134 AUGUCUAUCAGCGCAGCUACUGC 1674 135 UGUCUAUCAGCGCAGCUACUGCC 1675 136 GUCUAUCAGCGCAGCUACUGCCA 1676 137 UCUAUCAGCGCAGCUACUGCCAU 1677 138 CUAUCAGCGCAGCUACUGCCAUC 1678 139 UAUCAGCGCAGCUACUGCCAUCC 1679 140 AUCAGCGCAGCUACUGCCAUCCA 1680 141 UCAGCGCAGCUACUGCCAUCCAA 1681 142 CAGCGCAGCUACUGCCAUCCAAU 1682 143 AGCGCAGCUACUGCCAUCCAAUC 1683 144 GCGCAGCUACUGCCAUCCAAUCG 1684 145 CGCAGCUACUGCCAUCCAAUCGA 1685 146 GCAGCUACUGCCAUCCAAUCGAG 1686 147 CAGCUACUGCCAUCCAAUCGAGA 1687 148 AGCUACUGCCAUCCAAUCGAGAC 1688 149 GCUACUGCCAUCCAAUCGAGACC 1689 150 CUACUGCCAUCCAAUCGAGACCC 1690 151 UACUGCCAUCCAAUCGAGACCCU 1691 152 ACUGCCAUCCAAUCGAGACCCUG 1692 153 CUGCCAUCCAAUCGAGACCCUGG 1693 154 UGCCAUCCAAUCGAGACCCUGGU 1694 155 GCCAUCCAAUCGAGACCCUGGUG 1695 156 CCAUCCAAUCGAGACCCUGGUGG 1696 157 CAUCCAAUCGAGACCCUGGUGGA 1697 158 AUCCAAUCGAGACCCUGGUGGAC 1698 159 UCCAAUCGAGACCCUGGUGGACA 1699 160 CCAAUCGAGACCCUGGUGGACAU 1700 161 CAAUCGAGACCCUGGUGGACAUC 1701 162 AAUCGAGACCCUGGUGGACAUCU 1702 163 AUCGAGACCCUGGUGGACAUCUU 1703 164 UCGAGACCCUGGUGGACAUCUUC 1704 165 CGAGACCCUGGUGGACAUCUUCC 1705 166 GAGACCCUGGUGGACAUCUUCCA 1706 167 AGACCCUGGUGGACAUCUUCCAG 1707 168 GACCCUGGUGGACAUCUUCCAGG 1708 169 ACCCUGGUGGACAUCUUCCAGGA 1709 170 CCCUGGUGGACAUCUUCCAGGAG 1710 171 CCUGGUGGACAUCUUCCAGGAGU 1711 172 CUGGUGGACAUCUUCCAGGAGUA 1712 173 UGGUGGACAUCUUCCAGGAGUAC 1713 174 GGUGGACAUCUUCCAGGAGUACC 1714 175 GUGGACAUCUUCCAGGAGUACCC 1715 176 UGGACAUCUUCCAGGAGUACCCU 1716 177 GGACAUCUUCCAGGAGUACCCUG 1717 178 GACAUCUUCCAGGAGUACCCUGA 1718 179 ACAUCUUCCAGGAGUACCCUGAU 1719 180 CAUCUUCCAGGAGUACCCUGAUG 1720 181 AUCUUCCAGGAGUACCCUGAUGA 1721 182 UCUUCCAGGAGUACCCUGAUGAG 1722 183 CUUCCAGGAGUACCCUGAUGAGA 1723 184 UUCCAGGAGUACCCUGAUGAGAU 1724 185 UCCAGGAGUACCCUGAUGAGAUC 1725 186 CCAGGAGUACCCUGAUGAGAUCG 1726 187 CAGGAGUACCCUGAUGAGAUCGA 1727 188 AGGAGUACCCUGAUGAGAUCGAG 1728 189 GGAGUACCCUGAUGAGAUCGAGU 1729 190 GAGUACCCUGAUGAGAUCGAGUA 1730 191 AGUACCCUGAUGAGAUCGAGUAC 1731 192 GUACCCUGAUGAGAUCGAGUACA 1732 193 UACCCUGAUGAGAUCGAGUACAU 1733 194 ACCCUGAUGAGAUCGAGUACAUC 1734 195 CCCUGAUGAGAUCGAGUACAUCU 1735 196 CCUGAUGAGAUCGAGUACAUCUU 1736 197 CUGAUGAGAUCGAGUACAUCUUC 1737 198 UGAUGAGAUCGAGUACAUCUUCA 1738 199 GAUGAGAUCGAGUACAUCUUCAA 1739 200 AUGAGAUCGAGUACAUCUUCAAG 1740 201 UGAGAUCGAGUACAUCUUCAAGC 1741 202 GAGAUCGAGUACAUCUUCAAGCC 1742 203 AGAUCGAGUACAUCUUCAAGCCA 1743 204 GAUCGAGUACAUCUUCAAGCCAU 1744 205 AUCGAGUACAUCUUCAAGCCAUC 1745 206 UCGAGUACAUCUUCAAGCCAUCC 1746 207 CGAGUACAUCUUCAAGCCAUCCU 1747 208 GAGUACAUCUUCAAGCCAUCCUG 1748 209 AGUACAUCUUCAAGCCAUCCUGU 1749 210 GUACAUCUUCAAGCCAUCCUGUG 1750 211 UACAUCUUCAAGCCAUCCUGUGU 1751 212 ACAUCUUCAAGCCAUCCUGUGUG 1752 213 CAUCUUCAAGCCAUCCUGUGUGC 1753 214 AUCUUCAAGCCAUCCUGUGUGCC 1754 215 UCUUCAAGCCAUCCUGUGUGCCC 1755 216 CUUCAAGCCAUCCUGUGUGCCCC 1756 217 UUCAAGCCAUCCUGUGUGCCCCU 1757 218 UCAAGCCAUCCUGUGUGCCCCUG 1758 219 CAAGCCAUCCUGUGUGCCCCUGA 1759 220 AAGCCAUCCUGUGUGCCCCUGAU 1760 221 AGCCAUCCUGUGUGCCCCUGAUG 1761 222 GCCAUCCUGUGUGCCCCUGAUGC 1762 223 CCAUCCUGUGUGCCCCUGAUGCG 1763 224 CAUCCUGUGUGCCCCUGAUGCGA 1764 225 AUCCUGUGUGCCCCUGAUGCGAU 1765 226 UCCUGUGUGCCCCUGAUGCGAUG 1766 227 CCUGUGUGCCCCUGAUGCGAUGC 1767 228 CUGUGUGCCCCUGAUGCGAUGCG 1768 229 UGUGUGCCCCUGAUGCGAUGCGG 1769 230 GUGUGCCCCUGAUGCGAUGCGGG 1770 231 UGUGCCCCUGAUGCGAUGCGGGG 1771 232 GUGCCCCUGAUGCGAUGCGGGGG 1772 233 UGCCCCUGAUGCGAUGCGGGGGC 1773 234 GCCCCUGAUGCGAUGCGGGGGCU 1774 235 CCCCUGAUGCGAUGCGGGGGCUG 1775 236 CCCUGAUGCGAUGCGGGGGCUGC 1776 237 CCUGAUGCGAUGCGGGGGCUGCU 1777 238 CUGAUGCGAUGCGGGGGCUGCUG 1778 239 UGAUGCGAUGCGGGGGCUGCUGC 1779 240 GAUGCGAUGCGGGGGCUGCUGCA 1780 241 AUGCGAUGCGGGGGCUGCUGCAA 1781 242 UGCGAUGCGGGGGCUGCUGCAAU 1782 243 GCGAUGCGGGGGCUGCUGCAAUG 1783 244 CGAUGCGGGGGCUGCUGCAAUGA 1784 245 GAUGCGGGGGCUGCUGCAAUGAC 1785 246 AUGCGGGGGCUGCUGCAAUGACG 1786 247 UGCGGGGGCUGCUGCAAUGACGA 1787 248 GCGGGGGCUGCUGCAAUGACGAG 1788 249 CGGGGGCUGCUGCAAUGACGAGG 1789 250 GGGGGCUGCUGCAAUGACGAGGG 1790 251 GGGGCUGCUGCAAUGACGAGGGC 1791 252 GGGCUGCUGCAAUGACGAGGGCC 1792 253 GGCUGCUGCAAUGACGAGGGCCU 1793 254 GCUGCUGCAAUGACGAGGGCCUG 1794 255 CUGCUGCAAUGACGAGGGCCUGG 1795 256 UGCUGCAAUGACGAGGGCCUGGA 1796 257 GCUGCAAUGACGAGGGCCUGGAG 1797 258 CUGCAAUGACGAGGGCCUGGAGU 1798 259 UGCAAUGACGAGGGCCUGGAGUG 1799 260 GCAAUGACGAGGGCCUGGAGUGU 1800 261 CAAUGACGAGGGCCUGGAGUGUG 1801 262 AAUGACGAGGGCCUGGAGUGUGU 1802 263 AUGACGAGGGCCUGGAGUGUGUG 1803 264 UGACGAGGGCCUGGAGUGUGUGC 1804 265 GACGAGGGCCUGGAGUGUGUGCC 1805 266 ACGAGGGCCUGGAGUGUGUGCCC 1806 267 CGAGGGCCUGGAGUGUGUGCCCA 1807 268 GAGGGCCUGGAGUGUGUGCCCAC 1808 269 AGGGCCUGGAGUGUGUGCCCACU 1809 270 GGGCCUGGAGUGUGUGCCCACUG 1810 271 GGCCUGGAGUGUGUGCCCACUGA 1811 272 GCCUGGAGUGUGUGCCCACUGAG 1812 273 CCUGGAGUGUGUGCCCACUGAGG 1813 274 CUGGAGUGUGUGCCCACUGAGGA 1814 275 UGGAGUGUGUGCCCACUGAGGAG 1815 276 GGAGUGUGUGCCCACUGAGGAGU 1816 277 GAGUGUGUGCCCACUGAGGAGUC 1817 278 AGUGUGUGCCCACUGAGGAGUCC 1818 279 GUGUGUGCCCACUGAGGAGUCCA 1819 280 UGUGUGCCCACUGAGGAGUCCAA 1820 281 GUGUGCCCACUGAGGAGUCCAAC 1821 282 UGUGCCCACUGAGGAGUCCAACA 1822 283 GUGCCCACUGAGGAGUCCAACAU 1823 284 UGCCCACUGAGGAGUCCAACAUC 1824 285 GCCCACUGAGGAGUCCAACAUCA 1825 286 CCCACUGAGGAGUCCAACAUCAC 1826 287 CCACUGAGGAGUCCAACAUCACC 1827 288 CACUGAGGAGUCCAACAUCACCA 1828 289 ACUGAGGAGUCCAACAUCACCAU 1829 290 CUGAGGAGUCCAACAUCACCAUG 1830 291 UGAGGAGUCCAACAUCACCAUGC 1831 292 GAGGAGUCCAACAUCACCAUGCA 1832 293 AGGAGUCCAACAUCACCAUGCAG 1833 294 GGAGUCCAACAUCACCAUGCAGA 1834 295 GAGUCCAACAUCACCAUGCAGAU 1835 296 AGUCCAACAUCACCAUGCAGAUU 1836 297 GUCCAACAUCACCAUGCAGAUUA 1837 298 UCCAACAUCACCAUGCAGAUUAU 1838 299 CCAACAUCACCAUGCAGAUUAUG 1839 300 CAACAUCACCAUGCAGAUUAUGC 1840 301 AACAUCACCAUGCAGAUUAUGCG 1841 302 ACAUCACCAUGCAGAUUAUGCGG 1842 303 CAUCACCAUGCAGAUUAUGCGGA 1843 304 AUCACCAUGCAGAUUAUGCGGAU 1844 305 UCACCAUGCAGAUUAUGCGGAUC 1845 306 CACCAUGCAGAUUAUGCGGAUCA 1846 307 ACCAUGCAGAUUAUGCGGAUCAA 1847 308 CCAUGCAGAUUAUGCGGAUCAAA 1848 309 CAUGCAGAUUAUGCGGAUCAAAC 1849 310 AUGCAGAUUAUGCGGAUCAAACC 1850 311 UGCAGAUUAUGCGGAUCAAACCU 1851 312 GCAGAUUAUGCGGAUCAAACCUC 1852 313 CAGAUUAUGCGGAUCAAACCUCA 1853 314 AGAUUAUGCGGAUCAAACCUCAC 1854 315 GAUUAUGCGGAUCAAACCUCACC 1855 316 AUUAUGCGGAUCAAACCUCACCA 1856 317 UUAUGCGGAUCAAACCUCACCAA 1857 318 UAUGCGGAUCAAACCUCACCAAG 1858 319 AUGCGGAUCAAACCUCACCAAGG 1859 320 UGCGGAUCAAACCUCACCAAGGC 1860 321 GCGGAUCAAACCUCACCAAGGCC 1861 322 CGGAUCAAACCUCACCAAGGCCA 1862 323 GGAUCAAACCUCACCAAGGCCAG 1863 324 GAUCAAACCUCACCAAGGCCAGC 1864 325 AUCAAACCUCACCAAGGCCAGCA 1865 326 UCAAACCUCACCAAGGCCAGCAC 1866 327 CAAACCUCACCAAGGCCAGCACA 1867 328 AAACCUCACCAAGGCCAGCACAU 1868 329 AACCUCACCAAGGCCAGCACAUA 1869 330 ACCUCACCAAGGCCAGCACAUAG 1870 331 CCUCACCAAGGCCAGCACAUAGG 1871 332 CUCACCAAGGCCAGCACAUAGGA 1872 333 UCACCAAGGCCAGCACAUAGGAG 1873 334 CACCAAGGCCAGCACAUAGGAGA 1874 335 ACCAAGGCCAGCACAUAGGAGAG 1875 336 CCAAGGCCAGCACAUAGGAGAGA 1876 337 CAAGGCCAGCACAUAGGAGAGAU 1877 338 AAGGCCAGCACAUAGGAGAGAUG 1878 339 AGGCCAGCACAUAGGAGAGAUGA 1879 340 GGCCAGCACAUAGGAGAGAUGAG 1880 341 GCCAGCACAUAGGAGAGAUGAGC 1881 342 CCAGCACAUAGGAGAGAUGAGCU 1882 343 CAGCACAUAGGAGAGAUGAGCUU 1883 344 AGCACAUAGGAGAGAUGAGCUUC 1884 345 GCACAUAGGAGAGAUGAGCUUCC 1885 346 CACAUAGGAGAGAUGAGCUUCCU 1886 347 ACAUAGGAGAGAUGAGCUUCCUA 1887 348 CAUAGGAGAGAUGAGCUUCCUAC 1888 349 AUAGGAGAGAUGAGCUUCCUACA 1889 350 UAGGAGAGAUGAGCUUCCUACAG 1890 351 AGGAGAGAUGAGCUUCCUACAGC 1891 352 GGAGAGAUGAGCUUCCUACAGCA 1892 353 GAGAGAUGAGCUUCCUACAGCAC 1893 354 AGAGAUGAGCUUCCUACAGCACA 1894 355 GAGAUGAGCUUCCUACAGCACAA 1895 356 AGAUGAGCUUCCUACAGCACAAC 1896 357 GAUGAGCUUCCUACAGCACAACA 1897 358 AUGAGCUUCCUACAGCACAACAA 1898 359 UGAGCUUCCUACAGCACAACAAA 1899 360 GAGCUUCCUACAGCACAACAAAU 1900 361 AGCUUCCUACAGCACAACAAAUG 1901 362 GCUUCCUACAGCACAACAAAUGU 1902 363 CUUCCUACAGCACAACAAAUGUG 1903 364 UUCCUACAGCACAACAAAUGUGA 1904 365 UCCUACAGCACAACAAAUGUGAA 1905 366 CCUACAGCACAACAAAUGUGAAU 1906 367 CUACAGCACAACAAAUGUGAAUG 1907 368 UACAGCACAACAAAUGUGAAUGC 1908 369 ACAGCACAACAAAUGUGAAUGCA 1909 370 CAGCACAACAAAUGUGAAUGCAG 1910 371 AGCACAACAAAUGUGAAUGCAGA 1911 372 GCACAACAAAUGUGAAUGCAGAC 1912 373 CACAACAAAUGUGAAUGCAGACC 1913 374 ACAACAAAUGUGAAUGCAGACCA 1914 375 CAACAAAUGUGAAUGCAGACCAA 1915 376 AACAAAUGUGAAUGCAGACCAAA 1916 377 ACAAAUGUGAAUGCAGACCAAAG 1917 378 CAAAUGUGAAUGCAGACCAAAGA 1918 379 AAAUGUGAAUGCAGACCAAAGAA 1919 380 AAUGUGAAUGCAGACCAAAGAAA 1920 381 AUGUGAAUGCAGACCAAAGAAAG 1921 382 UGUGAAUGCAGACCAAAGAAAGA 1922 383 GUGAAUGCAGACCAAAGAAAGAU 1923 384 UGAAUGCAGACCAAAGAAAGAUA 1924 385 GAAUGCAGACCAAAGAAAGAUAG 1925 386 AAUGCAGACCAAAGAAAGAUAGA 1926 387 AUGCAGACCAAAGAAAGAUAGAG 1927 388 UGCAGACCAAAGAAAGAUAGAGC 1928 389 GCAGACCAAAGAAAGAUAGAGCA 1929 390 CAGACCAAAGAAAGAUAGAGCAA 1930 391 AGACCAAAGAAAGAUAGAGCAAG 1931 392 GACCAAAGAAAGAUAGAGCAAGA 1932 393 ACCAAAGAAAGAUAGAGCAAGAC 1933 394 CCAAAGAAAGAUAGAGCAAGACA 1934 395 CAAAGAAAGAUAGAGCAAGACAA 1935 396 AAAGAAAGAUAGAGCAAGACAAG 1936 397 AAGAAAGAUAGAGCAAGACAAGA 1937 398 AGAAAGAUAGAGCAAGACAAGAA 1938 399 GAAAGAUAGAGCAAGACAAGAAA 1939 400 AAAGAUAGAGCAAGACAAGAAAA

TABLE 4b VEGF targeted duplexes Strand: S = sense, AS = Antisense SEQ SEQ position ID Target sequence ID in ORF NO: (5′-3′) Duplex ID Strand NO: Strand Sequences   1 2184 AUGAACUUUCUGCUGUCUUGGGU AL-DP-4043 S 1940 5 GAACUUUCUGCUGUCUUGGGU 3 AS 1941 3 UACUUGAAAGACGACAGAACCCA 5  22 2185 GUGCAUUGGAGCCUUGCCUUGCU AL-DP-4077 S 1942 5 GCAUUGGAGCCUUGCCUUGCU 3 AS 1943 3 CACGUAACCUCGGAACGGAACGA 5  47 2186 UCUACCUCCACCAUGCCAAGUGG AL-DP-4021 S 1944 5 UACCUCCACCAUGCCAAGUTT 3 AS 1945 3 TTAUGGAGGUGGUACGGUUCA 5  48 2187 CUACCUCCACCAUGCCAAGUGGU AL-DP-4109 S 1946 5 ACCUCCACCAUGCCAAGUGTT 3 AS 1947 3 TTUGGAGGUGGUACGGUUCAC 5  50 2188 ACCUCCACCAUGCCAAGUGGUCC AL-DP-4006 S 1948 5 CUCCACCAUGCCAAGUGGUCC 3 AS 1949 3 UGGAGGUGGUACGGUUCACCAGG 5 AL-DP-4083 S 1950 5 CUCCACCAUGCCAAGUGGUTT 3 AS 1951 3 TTGAGGUGGUACGGUUCACCA 5  51 2189 CCUCCACCAUGCCAAGUGGUCCC AL-DP-4047 S 1952 5 UCCACCAUGCCAAGUGGUCCC 3 AS 1953 3 GGAGGUGGUACGGUUCACCAGGG 5 AL-DP-4017 S 1954 5 UCCACCAUGCCAAGUGGUCTT 3 AS 1955 3 TTAGGUGGUACGGUUCACCAG 5  52 2190 CUCCACCAUGCCAAGUGGUCCCA AL-DP-4048 S 1956 5 CCACCAUGCCAAGUGGUCCCA 3 AS 1957 3 GAGGUGGUACGGUUCACCAGGGU 5 AL-DP-4103 S 1958 5 CCACCAUGCCAAGUGGUCCTT 3 AS 1959 3 TTGGUGGUACGGUUCACCAGG 5  53 2191 UCCACCAUGCCAAGUGGUCCCAG AL-DP-4035 S 1960 5 CACCAUGCCAAGUGGUCCCAG 3 AS 1961 3 AGGUGGUACGGUUCACCAGGGUC 5 AL-DP-4018 S 1962 5 CACCAUGCCAAGUGGUCCCTT 3 AS 1963 3 TTGUGGUACGGUUCACCAGGG 5  54 2192 CCACCAUGCCAAGUGGUCCCAGG AL-DP-4036 S 1964 5 ACCAUGCCAAGUGGUCCCAGG 3 AS 1965 3 GGUGGUACGGUUCACCAGGGUCC 5 AL-DP-4084 S 1966 5 ACCAUGCCAAGUGGUCCCATT 3 AS 1967 3 TTUGGUACGGUUCACCAGGGU 5  55 2193 CACCAUGCCAAGUGGUCCCAGGC AL-DP-4093 S 1968 5 CCAUGCCAAGUGGUCCCAGGC 3 AS 1969 3 GUGGUACGGUUCACCAGGGUCCG 5 AL-DP-4085 S 1970 5 CCAUGCCAAGUGGUCCCAGTT 3 AS 1971 3 TTGGUACGGUUCACCAGGGUC 5  56 2194 ACCAUGCCAAGUGGUCCCAGGCU AL-DP-4037 S 1972 5 CAUGCCAAGUGGUCCCAGGCU 3 AS 1973 3 UGGUACGGUUCACCAGGGUCCGA 5 AL-DP-4054 S 1974 5 CAUGCCAAGUGGUCCCAGGTT 3 AS 1975 3 TTGUACGGUUCACCAGGGUCC 5  57 2195 CCAUGCCAAGUGGUCCCAGGCUG AL-DP-4038 S 1976 5 AUGCCAAGUGGUCCCAGGCUG 3 AS 1977 3 GGUACGGUUCACCAGGGUCCGAC 5 AL-DP-4086 S 1978 5 AUGCCAAGUGGUCCCAGGCTT 3 AS 1979 3 TTUACGGUUCACCAGGGUCCG 5  58 2196 CAUGCCAAGUGGUCCCAGGCUGC AL-DP-4049 S 1980 5 UGCCAAGUGGUCCCAGGCUGC 3 AS 1981 3 GUACGGUUCACCAGGGUCCGACG 5 AL-DP-4087 S 1982 5 UGCCAAGUGGUCCCAGGCUTT 3 AS 1983 3 TTACGGUUCACCAGGGUCCGA 5  59 2197 AUGCCAAGUGGUCCCAGGCUGCA AL-DP-4001 S 1984 5 GCCAAGUGGUCCCAGGCUGCA 3 AS 1985 3 UACGGUUCACCAGGGUCCGACGU 5 AL-DP-4052 A 1986 5 GCCAAGUGGUCCCAGGCUGTT 3 AS 1987 3 TTCGGUUCACCAGGGUCCGAC 5  60 2198 UGCCAAGUGGUCCCAGGCUGCAC AL-DP-4007 S 1988 5 CCAAGUGGUCCCAGGCUGCAC 3 AS 1989 3 ACGGUUCACCAGGGUCCGACGUG 5 AL-DP-4088 S 1990 5 CCAAGUGGUCCCAGGCUGCTT 3 AS 1991 3 TTGGUUCACCAGGGUCCGACG 5  61 2199 GCCAAGUGGUCCCAGGCUGCACC AL-DP-4070 S 1992 5 CAAGUGGUCCCAGGCUGCACC 3 AS 1993 3 CGGUUCACCAGGGUCCGACGUGG 5 AL-DP-4055 S 1994 5 CAAGUGGUCCCAGGCUGCATT 3 AS 1995 3 TTGUUCACCAGGGUCCGACGU 5  62 2200 CCAAGUGGUCCCAGGCUGCACCC AL-DP-4071 S 1996 5 AAGUGGUCCCAGGCUGCACCC 3 AS 1997 3 GGUUCACCAGGGUCCGACGUGGG 5 AL-DP-4056 S 1998 5 AAGUGGUCCCAGGCUGCACTT 3 AS 1999 3 TTUUCACCAGGGUCCGACGUG 5  63 2201 CAAGUGGUCCCAGGCUGCACCCA AL-DP-4072 S 2000 5 AGUGGUCCCAGGCUGCACCCA 3 AS 2001 3 GUUCACCAGGGUCCGACGUGGGU 5 AL-DP-4057 S 2002 5 AGUGGUCCCAGGCUGCACCTT 3 AS 2003 3 TTUCACCAGGGUCCGACGUGG 5  64 2202 AAGUGGUCCCAGGCUGCACCCAU AL-DP-4066 S 2004 5 GUGGUCCCAGGCUGCACCCTT 3 AS 2005 3 TTCACCAGGGUCCGACGUGGG 5  99 2203 AGGGCAGAAUCAUCACGAAGUGG AL-DP-4022 S 2006 5 GGCAGAAUCAUCACGAAGUTT 3 AS 2007 3 TTCCGUCUUAGUAGUGCUUCA 5 100 2204 GGGCAGAAUCAUCACGAAGUGGU AL-DP-4023 S 2008 5 GCAGAAUCAUCACGAAGUGTT 3 AS 2009 3 TTCGUCUUAGUAGUGCUUCAC 5 101 2205 GGCAGAAUCAUCACGAAGUGGUG AL-DP-4024 S 2010 5 CAGAAUCAUCACGAAGUGGTT 3 AS 2011 3 TTGUCUUAGUAGUGCUUCACC 5 102 2206 GCAGAAUCAUCACGAAGUGGUGA AL-DP-4076 S 2012 5 AGAAUCAUCACGAAGUGGUGA 3 AS 2013 3 CGUCUUAGUAGUGCUUCACCACU 5 AL-DP-4019 S 2014 5 AGAAUCAUCACGAAGUGGUTT 3 AS 2015 3 TTUCUUAGUAGUGCUUCACCA 5 103 2207 CAGAAUCAUCACGAAGUGGUGAA AL-DP-4025 S 2016 5 GAAUCAUCACGAAGUGGUGTT 3 AS 2017 3 TTCUUAGUAGUGCUUCACCAC 5 104 2208 AGAAUCAUCACGAAGUGGUGAAG AL-DP-4110 S 2018 5 AAUCAUCACGAAGUGGUGATT 3 AS 2019 3 TTUUAGUAGUGCUUCACCACU 5 105 2209 GAAUCAUCACGAAGUGGUGAAGU AL-DP-4068 S 2020 5 AUCAUCACGAAGUGGUGAATT 3 AS 2021 3 TTUAGUAGUGCUUCACCACUU 5 113 2210 ACGAAGUGGUGAAGUUCAUGGAU AL-DP-4078 S 2022 5 GAAGUGGUGAAGUUCAUGGAU 3 AS 2023 3 UGCUUCACCACUUCAAGUACCUA 5 121 2211 GUGAAGUUCAUGGAUGUCUAUCA AL-DP-4080 S 2024 5 GAAGUUCAUGGAUGUCUAUCA 3 AS 2025 3 CACUUCAAGUACCUACAGAUAGU 5 129 2212 CAUGGAUGUCUAUCAGCGCAGCU AL-DP-4111 S 2026 5 UGGAUGUCUAUCAGCGCAGTT 3 AS 2027 3 TTACCUACAGAUAGUCGCGUC 5 130 2213 AUGGAUGUCUAUCAGCGCAGCUA AL-DP-4041 S 2028 5 GGAUGUCUAUCAGCGCAGCUA 3 AS 2029 3 UACCUACAGAUAGUCGCGUCGAU 5 AL-DP-4062 S 2030 5 GGAUGUCUAUCAGCGCAGCTT 3 AS 2031 3 TTCCUACAGAUAGUCGCGUCG 5 131 2214 UGGAUGUCUAUCAGCGCAGCUAC AL-DP-4069 S 2032 5 GAUGUCUAUCAGCGCAGCUTT 3 AS 2033 3 TTCUACAGAUAGUCGCGUCGA 5 132 2215 GGAUGUCUAUCAGCGCAGCUACU AL-DP-4112 S 2034 5 AUGUCUAUCAGCGCAGCUATT 3 AS 2035 3 TTUACAGAUAGUCGCGUCGAU 5 133 2216 GAUGUCUAUCAGCGCAGCUACUG AL-DP-4026 S 2036 5 UGUCUAUCAGCGCAGCUACTT 3 AS 2037 3 TTACAGAUAGUCGCGUCGAUG 5 134 2217 AUGUCUAUCAGCGCAGCUACUGC AL-DP-4095 S 2038 5 GUCUAUCAGCGCAGCUACUGC 3 AS 2039 3 UACAGAUAGUCGCGUCGAUGACG 5 AL-DP-4020 S 2040 5 GUCUAUCAGCGCAGCUACUTT 3 AS 2041 3 TTCAGAUAGUCGCGUCGAUGA 5 135 2218 UGUCUAUCAGCGCAGCUACUGCC AL-DP-4027 S 2042 5 UCUAUCAGCGCAGCUACUGTT 3 AS 2043 3 TTAGAUAGUCGCGUCGAUGAC 5 144 2219 GCGCAGCUACUGCCAUCCAAUCG AL-DP-4081 S 2044 5 GCAGCUACUGCCAUCCAAUCG 3 AS 2045 3 CGCGUCGAUGACGGUAGGUUAGC 5 146 2220 GCAGCUACUGCCAUCCAAUCGAG AL-DP-4098 S 2046 5 AGCUACUGCCAUCCAAUCGAG 3 AS 2047 3 CGUCGAUGACGGUAGGUUAGCUC 5 149 2221 GCUACUGCCAUCCAAUCGAGACC AL-DP-4028 S 2048 5 UACUGCCAUCCAAUCGAGATT 3 AS 2049 3 TTAUGACGGUAGGUUAGCUCU 5 150 2222 CUACUGCCAUCCAAUCGAGACCC AL-DP-4029 S 2050 5 ACUGCCAUCCAAUCGAGACTT 3 AS 2051 3 TTUGACGGUAGGUUAGCUCUG 5 151 2223 UACUGCCAUCCAAUCGAGACCCU AL-DP-4030 S 2052 5 CUGCCAUCCAAUCGAGACCTT 3 AS 2053 3 TTGACGGUAGGUUAGCUCUGG 5 152 2224 ACUGCCAUCCAAUCGAGACCCUG AL-DP-4031 S 2054 5 UGCCAUCCAAUCGAGACCCTT 3 AS 2055 3 TTACGGUAGGUUAGCUCUGGG 5 166 2225 GAGACCCUGGUGGACAUCUUCCA AL-DP-4008 S 2056 5 GACCCUGGUGGACAUCUUCCA 3 AS 2057 3 CUCUGGGACCACCUGUAGAAGGU 5 AL-DP-4058 S 2058 5 GACCCUGGUGGACAUCUUCTT 3 AS 2059 3 TTCUGGGACCACCUGUAGAAG 5 167 2226 AGACCCUGGUGGACAUCUUCCAG AL-DP-4009 S 2060 5 ACCCUGGUGGACAUCUUCCAG 3 AS 2061 3 UCUGGGACCACCUGUAGAAGGUC 5 AL-DP-4059 S 2062 5 ACCCUGGUGGACAUCUUCCTT 3 AS 2063 3 TTUGGGACCACCUGUAGAAGG 5 168 2227 GACCCUGGUGGACAUCUUCCAGG AL-DP-4010 S 2064 5 CCCUGGUGGACAUCUUCCAGG 3 AS 2065 3 CUGGGACCACCUGUAGAAGGUCC 5 AL-DP-4060 S 2066 5 CCCUGGUGGACAUCUUCCATT 3 AS 2067 3 TTGGGACCACCUGUAGAAGGU 5 169 2228 ACCCUGGUGGACAUCUUCCAGGA AL-DP-4073 S 2068 5 CCUGGUGGACAUCUUCCAGGA 3 AS 2069 3 UGGGACCACCUGUAGAAGGUCCU 5 AL-DP-4104 S 2070 5 CCUGGUGGACAUCUUCCAGTT 3 AS 2071 3 TTGGACCACCUGUAGAAGGUC 5 170 2229 CCCUGGUGGACAUCUUCCAGGAG AL-DP-4011 S 2072 5 CUGGUGGACAUCUUCCAGGAG 3 AS 2073 3 GGGACCACCUGUAGAAGGUCCUC 5 AL-DP-4089 S 2074 5 CUGGUGGACAUCUUCCAGGTT 3 AS 2075 3 TTGACCACCUGUAGAAGGUCC 5 171 2230 CCUGGUGGACAUCUUCCAGGAGU AL-DP-4074 S 2076 5 UGGUGGACAUCUUCCAGGAGU 3 AS 2077 3 GGACCACCUGUAGAAGGUCCUCA 5 AL-DP-4090 S 2078 5 UGGUGGACAUCUUCCAGGATT 3 AS 2079 3 TTACCACCUGUAGAAGGUCCU 5 172 2231 CUGGUGGACAUCUUCCAGGAGUA AL-DP-4039 S 2080 5 GGUGGACAUCUUCCAGGAGUA 3 AS 2081 3 GACCACCUGUAGAAGGUCCUCAU 5 AL-DP-4091 S 2082 5 GGUGGACAUCUUCCAGGAGTT 3 AS 2083 3 TTCCACCUGUAGAAGGUCCUC 5 175 2232 GUGGACAUCUUCCAGGAGUACCC AL-DP-4003 S 2084 5 GGACAUCUUCCAGGAGUACCC 3 AS 2085 3 CCUGUAGAAGGUCCUCAUGGG 5 AL-DP-4116 S 2086 5 GGACAUCUUCCAGGAGUACCC 3 AS 2087 3 CCUGUAGAAGGUCCUCAUGGG 5 AL-DP-4015 S 2088 5 GGACAUCUUCCAGGAGUACTT 3 AS 2089 3 TTCCUGUAGAAGGUCCUCAUG 5 AL-DP-4120 S 2090 5 GGACAUCUUCCAGGAGUAC 3 AS 2091 3 CCUGUAGAAGGUCCUCAUG 5 179 2233 ACAUCUUCCAGGAGUACCCUGAU AL-DP-4099 S 2092 5 AUCUUCCAGGAGUACCCUGAU 3 AS 2093 3 UGUAGAAGGUCCUCAUGGGACUA 5 191 2234 AGUACCCUGAUGAGAUCGAGUAC AL-DP-4032 S 2094 5 UACCCUGAUGAGAUCGAGUTT 3 AS 2095 3 TTAUGGGACUACUCUAGCUCA 5 192 2235 GUACCCUGAUGAGAUCGAGUACA AL-DP-4042 S 2096 5 ACCCUGAUGAGAUCGAGUACA 3 AS 2097 3 CAUGGGACUACUCUAGCUCAUGU 5 AL-DP-4063 S 2098 5 ACCCUGAUGAGAUCGAGUATT 3 AS 2099 3 TTUGGGACUACUCUAGCUCAU 5 209 2236 AGUACAUCUUCAAGCCAUCCUGU AL-DP-4064 S 2100 5 UACAUCUUCAAGCCAUCCUTT 3 AS 2101 3 TTAUGUAGAAGUUCGGUAGGA 5 260 2237 GCAAUGACGAGGGCCUGGAGUGU AL-DP-4044 S 2102 5 AAUGACGAGGGCCUGGAGUGU 3 AS 2103 3 CGUUACUGCUCCCGGACCUCACA 5 263 2238 AUGACGAGGGCCUGGAGUGUGUG AL-DP-4045 S 2104 5 GACGAGGGCCUGGAGUGUGUG 3 AS 2105 3 UACUGCUCCCGGACCUCACACAC 5 279 2239 GUGUGUGCCCACUGAGGAGUCCA AL-DP-4046 S 2106 5 GUGUGCCCACUGAGGAGUCCA 3 AS 2107 3 CACACACGGGUGACUCCUCAGGU 5 281 2240 GUGUGCCCACUGAGGAGUCCAAC AL-DP-4096 S 2108 5 GUGCCCACUGAGGAGUCCAAC 3 AS 2109 3 CACACGGGUGACUCCUCAGGUUG 5 283 2241 GUGCCCACUGAGGAGUCCAACAU AL-DP-4040 S 2110 5 GCCCACUGAGGAGUCCAACAU 3 AS 2111 3 CACGGGUGACUCCUCAGGUUGUA 5 289 2242 ACUGAGGAGUCCAACAUCACCAU AL-DP-4065 S 2112 5 UGAGGAGUCCAACAUCACCTT 3 AS 2113 3 TTACUCCUCAGGUUGUAGUGG 5 302 2243 ACAUCACCAUGCAGAUUAUGCGG AL-DP-4100 S 2114 5 AUCACCAUGCAGAUUAUGCGG 3 AS 2115 3 UGUAGUGGUACGUCUAAUACGCC 5 305 2244 UCACCAUGCAGAUUAUGCGGAUC AL-DP-4033 S 2116 5 ACCAUGCAGAUUAUGCGGATT 3 AS 2117 3 TTUGGUACGUCUAAUACGCCU 5 310 2245 AUGCAGAUUAUGCGGAUCAAACC AL-DP-4101 S 2118 5 GCAGAUUAUGCGGAUCAAACC 3 AS 2119 3 UACGUCUAAUACGCCUAGUUUGG 5 312 2246 GCAGAUUAUGCGGAUCAAACCUC AL-DP-4102 S 2120 5 AGAUUAUGCGGAUCAAACCUC 3 AS 2121 3 CGUCUAAUACGCCUAGUUUGGAG 5 315 2247 GAUUAUGCGGAUCAAACCUCACC AL-DP-4034 S 2122 5 UUAUGCGGAUCAAACCUCATT 3 AS 2123 3 TTAAUACGCCUAGUUUGGAGU 5 316 2248 AUUAUGCGGAUCAAACCUCACCA AL-DP-4113 S 2124 5 UAUGCGGAUCAAACCUCACTT 3 AS 2125 3 TTAUACGCCUAGUUUGGAGUG 5 317 2249 UUAUGCGGAUCAAACCUCACCAA AL-DP-4114 S 2126 5 AUGCGGAUCAAACCUCACCTT 3 AS 2127 3 TTUACGCCUAGUUUGGAGUGG 5 319 2250 AUGCGGAUCAAACCUCACCAAGG AL-DP-4002 S 2128 5 GCGGAUCAAACCUCACCAAGG 3 AS 2129 3 UACGCCUAGUUUGGAGUGGUUCC 5 AL-DP-4115 S 2130 5 GCGGAUCAAACCUCACCAA 3 AS 2131 3 CGCCUAGUUUGGAGUGGUU 5 AL-DP-4014 S 2132 5 GCGGAUCAAACCUCACCAATT 3 AS 2133 3 TTCGCCUAGUUUGGAGUGGUU 5 AL-DP-4119 S 2134 5 GCGGAUCAAACCUCACCAA 3 AS 2135 3 CGCCUAGUUUGGAGUGGUU 5 321 2251 GCGGAUCAAACCUCACCAAGGCC AL-DP-4013 S 2136 5 GGAUCAAACCUCACCAAGGCC 3 AS 2137 3 CGCCUAGUUUGGAGUGGUUCCGG 5 341 2252 GCCAGCACAUAGGAGAGAUGAGC AL-DP-4075 S 2138 5 CAGCACAUAGGAGAGAUGAGC 3 AS 2139 3 CGGUCGUGUAUCCUCUCUACUCG 5 AL-DP-4105 S 2140 5 CAGCACAUAGGAGAGAUGATT 3 AS 2141 3 TTGUCGUGUAUCCUCUCUACU 5 342 2253 CCAGCACAUAGGAGAGAUGAGCU AL-DP-4050 S 2142 5 AGCACAUAGGAGAGAUGAGCU 3 AS 2143 3 GGUCGUGUAUCCUCUCUACUCGA 5 AL-DP-4106 S 2144 5 AGCACAUAGGAGAGAUGAGTT 3 AS 2145 3 TTUCGUGUAUCCUCUCUACUC 5 343 2254 CAGCACAUAGGAGAGAUGAGCUU AL-DP-4094 S 2146 5 GCACAUAGGAGAGAUGAGCUU 3 AS 2147 3 GUCGUGUAUCCUCUCUACUCGAA 5 AL-DP-4118 S 2148 5 GCACAUAGGAGAGAUGAGCUU 3 AS 2149 3 CGUGUAUCCUCUCUACUCGAA 5 AL-DP-4107 S 2150 5 GCACAUAGGAGAGAUGAGCTT 3 AS 2151 3 TTCGUGUAUCCUCUCUACUCG 5 AL-DP-4122 S 2152 5 GCACAUAGGAGAGAUGAGC 3 AS 2153 3 CGUGUAUCCUCUCUACUCG 5 344 2255 AGCACAUAGGAGAGAUGAGCUUC AL-DP-4012 S 2154 5 CACAUAGGAGAGAUGAGCUUC 3 AS 2155 3 UCGUGUAUCCUCUCUACUCGAAG 5 AL-DP-4108 S 2156 5 CACAUAGGAGAGAUGAGCUTT 3 AS 2157 3 TTGUGUAUCCUCUCUACUCGA 5 346 2256 CACAUAGGAGAGAUGAGCUUCCU AL-DP-4051 S 2158 5 CAUAGGAGAGAUGAGCUUCCU 3 AS 2159 3 GUGUAUCCUCUCUACUCGAAGGA 5 AL-DP-4061 S 2160 5 CAUAGGAGAGAUGAGCUUCTT 3 AS 2161 3 TTGUAUCCUCUCUACUCGAAG 5 349 2257 AUAGGAGAGAUGAGCUUCCUACA AL-DP-4082 S 2162 5 AGGAGAGAUGAGCUUCCUACA 3 AS 2163 3 UAUCCUCUCUACUCGAAGGAUGU 5 369 2258 ACAGCACAACAAAUGUGAAUGCA AL-DP-4079 S 2164 5 AGCACAACAAAUGUGAAUGCA 3 AS 2165 3 UGUCGUGUUGUUUACACUUACGU 5 372 2259 GCACAACAAAUGUGAAUGCAGAC AL-DP-4097 S 2166 5 ACAACAAAUGUGAAUGCAGAC 3 AS 2167 3 CGUGUUGUUUACACUUACGUCUG 5 379 2260 AAAUGUGAAUGCAGACCAAAGAA AL-DP-4067 S 2168 5 AUGUGAAUGCAGACCAAAGTT 3 AS 2169 3 TTUACACUUACGUCUGGUUUC 5 380 2261 AAUGUGAAUGCAGACCAAAGAAA AL-DP-4092 S 2170 5 UGUGAAUGCAGACCAAAGATT 3 AS 2171 3 TTACACUUACGUCUGGUUUCU 5 381 2262 AUGUGAAUGCAGACCAAAGAAAG AL-DP-4004 S 2172 5 GUGAAUGCAGACCAAAGAAAG 3 AS 2173 3 UACACUUACGUCUGGUUUCUUUC 5 AL-DP-4117 S 2174 5 GUGAAUGCAGACCAAAGAAAG 3 AS 2175 3 CACUUACGUCUGGUUUCUUUC 5 AL-DP-4016 S 2176 5 GUGAAUGCAGACCAAAGAATT 3 AS 2177 3 TTCACUUACGUCUGGUUUCUU 5 AL-DP-4121 S 2178 5 GUGAAUGCAGACCAAAGAA 3 AS 2179 3 CACUUACGUCUGGUUUCUU 5 383 2263 GUGAAUGCAGACCAAAGAAAGAU AL-DP-4005 S 2180 5 GAAUGCAGACCAAAGAAAGAU 3 AS 2181 3 CACUUACGUCUGGUUUCUUUCUA 5 AL-DP-4053 S 2182 5 GAAUGCAGACCAAAGAAAGTT 3 AS 2183 3 TTCUUACGUCUGGUUUCUUUC 5

Example 2 Eg5 siRNA In Vitro Screening Via Cell Proliferation

As silencing of Eg5 has been shown to cause mitotic arrest (Weil, D, et al [2002] Biotechniques 33: 1244-8), a cell viability assay was used for siRNA activity screening. HeLa cells (14000 per well [Screens 1 and 3] or 10000 per well [Screen2])) were seeded in 96-well plates and simultaneously transfected with Lipofectamine 2000 (Invitrogen) at a final siRNA concentration in the well of 30 nM and at final concentrations of 50 nM (1^(st) screen) and 25 nM (2^(nd) screen). A subset of duplexes was tested at 25 nM in a third screen (Table 5).

Seventy-two hours post-transfection, cell proliferation was assayed the addition of WST-1 reagent (Roche) to the culture medium, and subsequent absorbance measurement at 450 nm. The absorbance value for control (non-transfected) cells was considered 100 percent, and absorbances for the siRNA transfected wells were compared to the control value. Assays were performed in sextuplicate for each of three screens. A subset of the siRNAs was further tested at a range of siRNA concentrations. Assays were performed in HeLa cells (14000 per well; method same as above, Table 5).

TABLE 5 Effects of Eg5 targeted duplexes on cell viability at 25 nM. Relative absorbance at 450 nm Screen I Screen II Screen III Duplex mean sd Mean sd mean Sd AL-DP-6226 20 10 28 11 43 9 AL-DP-6227 66 27 96 41 108 33 AL-DP-6228 56 28 76 22 78 18 AL-DP-6229 17 3 31 9 48 13 AL-DP-6230 48 8 75 11 73 7 AL-DP-6231 8 1 21 4 41 10 AL-DP-6232 16 2 37 7 52 14 AL-DP-6233 31 9 37 6 49 12 AL-DP-6234 103 40 141 29 164 45 AL-DP-6235 107 34 140 27 195 75 AL-DP-6236 48 12 54 12 56 12 AL-DP-6237 73 14 108 18 154 37 AL-DP-6238 64 9 103 10 105 24 AL-DP-6239 9 1 20 4 31 11 AL-DP-6240 99 7 139 16 194 43 AL-DP-6241 43 9 54 12 66 19 AL-DP-6242 6 1 15 7 36 8 AL-DP-6243 7 2 19 5 33 13 AL-DP-6244 7 2 19 3 37 13 AL-DP-6245 25 4 45 10 58 9 AL-DP-6246 34 8 65 10 66 13 AL-DP-6247 53 6 78 14 105 20 AL-DP-6248 7 0 22 7 39 12 AL-DP-6249 36 8 48 13 61 7

The nine siRNA duplexes that showed the greatest growth inhibition in Table 5 were re-tested at a range of siRNA concentrations in HeLa cells. The siRNA concentrations tested were 100 nM, 33.3 nM, 11.1 nM, 3.70 nM, 1.23 nM, 0.41 nM, 0.14 nM and 0.046 nM. Assays were performed in sextuplicate, and the concentration of each siRNA resulting in fifty percent inhibition of cell proliferation (IC₅₀) was calculated. This dose-response analysis was performed between two and four times for each duplex. Mean IC₅₀ values (nM) are given in Table 6.

TABLE 6 IC50 of siRNA: cell proliferation in HeLa cells Duplex Mean IC₅₀ AL-DP-6226 15.5 AL-DP-6229 3.4 AL-DP-6231 4.2 AL-DP-6232 17.5 AL-DP-6239 4.4 AL-DP-6242 5.2 AL-DP-6243 2.6 AL-DP-6244 8.3 AL-DP-6248 1.9

Example 3 Eg5 siRNA In Vitro Screening Via mRNA Inhibition

Directly before transfection, HeLa S3 (ATCC-Number: CCL-2.2, LCG Promochem GmbH, Wesel, Germany) cells were seeded at 1.5×10⁴ cells/well on 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) in 75 μl of growth medium (Ham's F12, 10% fetal calf serum, 100 u penicillin/100 μg/ml streptomycin, all from Bookroom AG, Berlin, Germany). Transfections were performed in quadruplicates. For each well 0.5 μl Lipofectamine2000 (Invitrogen GmbH, Karlsruhe, Germany) were mixed with 12 μl Opti-MEM (Invitrogen) and incubated for 15 min at room temperature. For the siRNA concentration being 50 nM in the 100 μl transfection volume, 1 μl of a 5 μM siRNA were mixed with 11.5 μl Opti-MEM per well, combined with the Lipofectamine2000-Opti-MEM mixture and again incubated for 15 minutes at room temperature. siRNA-Lipofectamine2000-complexes were applied completely (25 μl each per well) to the cells and cells were incubated for 24 h at 37° C. and 5% CO₂ in a humidified incubator (Heroes GmbH, Hanau). The single dose screen was done once at 50 nM and at 25 nM, respectively.

Cells were harvested by applying 50 μl of lysis mixture (content of the QuantiGene bDNA-kit from Genospectra, Fremont, USA) to each well containing 100 μl of growth medium and were lysed at 53° C. for 30 min. Afterwards, 50 μl of the lists were incubated with probesets specific to human Eg5 and human GAPDH and proceeded according to the manufacturer's protocol for QuantiGene. In the end chemoluminescence was measured in a Victor2-Light (Perkin Elmer, Wiesbaden, Germany) as RLUs (relative light units) and values obtained with the hEg5 probeset were normalized to the respective GAPDH values for each well. Values obtained with siRNAs directed against Eg5 were related to the value obtained with an unspecific siRNA (directed against HCV) which was set to 100% (Tables 1b, 2b and 3b).

Effective siRNAs from the screen were further characterized by dose response curves. Transfections of dose response curves were performed at the following concentrations: 100 nM, 16.7 nM, 2.8 nM, 0.46 nM, 77 picoM, 12.8 picoM, 2.1 picoM, 0.35 picoM, 59.5 fM, 9.9 fM and mock (no siRNA) and diluted with Opti-MEM to a final concentration of 12.5 μl according to the above protocol. Data analysis was performed by using the Microsoft Excel add-in software XL-fit 4.2 (IDBS, Guildford, Surrey, UK) and applying the dose response model number 205 (Tables 1b, 2b and 3b).

The lead siRNA AD12115 was additionally analyzed by applying the WST-proliferation assay from Roche (as previously described).

A subset of 34 duplexes from Table 2 that showed greatest activity was assayed by transfection in HeLa cells at final concentrations ranging from 100 nM to 10 fM. Transfections were performed in quadruplicate. Two dose-response assays were performed for each duplex. The concentration giving 20% (IC20), 50% (ICSO) and 80% (IC80) reduction of KSP mRNA was calculated for each duplex (Table 7).

TABLE 7 Dose response mRNA inhibition of Eg5/KSP duplexes in HeLa cells Concentrations given in pM IC20s IC50s IC80s Duplex 1^(st) 2^(nd) 1st 2nd 1st 2nd name screen screen screen screen screen screen AD12077 1.19 0.80 6.14 10.16 38.63 76.16 AD12078 25.43 25.43 156.18 156.18 ND ND AD12085 9.08 1.24 40.57 8.52 257.68 81.26 AD12095 1.03 0.97 9.84 4.94 90.31 60.47 AD12113 4.00 5.94 17.18 28.14 490.83 441.30 AD12115 0.60 0.41 3.79 3.39 23.45 23.45 AD12125 31.21 22.02 184.28 166.15 896.85 1008.11 AD12134 2.59 5.51 17.87 22.00 116.36 107.03 AD12149 0.72 0.50 4.51 3.91 30.29 40.89 AD12151 0.53 6.84 4.27 10.72 22.88 43.01 AD12152 155.45 7.56 867.36 66.69 13165.27 ND AD12157 0.30 26.23 14.60 92.08 14399.22 693.31 AD12166 0.20 0.93 3.71 3.86 46.28 20.59 AD12180 28.85 28.85 101.06 101.06 847.21 847.21 AD12185 2.60 0.42 15.55 13.91 109.80 120.63 AD12194 2.08 1.11 5.37 5.09 53.03 30.92 AD12211 5.27 4.52 11.73 18.93 26.74 191.07 AD12257 4.56 5.20 21.68 22.75 124.69 135.82 AD12280 2.37 4.53 6.89 20.23 64.80 104.82 AD12281 8.81 8.65 19.68 42.89 119.01 356.08 AD12282 7.71 456.42 20.09 558.00 ND ND AD12285 ND 1.28 57.30 7.31 261.79 42.53 AD12292 40.23 12.00 929.11 109.10 ND ND AD12252 0.02 18.63 6.35 68.24 138.09 404.91 AD12275 25.76 25.04 123.89 133.10 1054.54 776.25 AD12266 4.85 7.80 10.00 32.94 41.67 162.65 AD12267 1.39 1.21 12.00 4.67 283.03 51.12 AD12264 0.92 2.07 8.56 15.12 56.36 196.78 AD12268 2.29 3.67 22.16 25.64 258.27 150.84 AD12279 1.11 28.54 23.19 96.87 327.28 607.27 AD12256 7.20 33.52 46.49 138.04 775.54 1076.76 AD12259 2.16 8.31 8.96 40.12 50.05 219.42 AD12276 19.49 6.14 89.60 59.60 672.51 736.72 AD12321 4.67 4.91 24.88 19.43 139.50 89.49 (ND—not determined)

Example 4 Silencing of Liver Eg5/KSP in Juvenile Rats Following Single-Bolus Administration of LNP01 Formulated siRNA

From birth until approximately 23 days of age, Eg5/KSP expression can be detected in the growing rat liver. Target silencing with a formulated Eg5/KSP siRNA was evaluated in juvenile rats using duplex AD-6248.

KSP Duplex Tested

Duplex ID Target Sense Antisense AD6248 KSP AccGAAGuGuuGuuu GGAcAAAcAAcACUUC GuccTsT GGUTsT (SEQ ID NO: 1238) (SEQ ID NO: 1239)

Methods

Dosing of Animals.

Male, juvenile Sprague-Dawley rats (19 days old) were administered single doses of lipidoid (“LNP01”) formulated siRNA via tail vein injection. Groups of ten animals received doses of 10 milligrams per kilogram (mg/kg) bodyweight of either AD6248 or an unspecific siRNA. Dose level refers to the amount of siRNA duplex administered in the formulation. A third group received phosphate-buffered saline. Animals were sacrificed two days after siRNA administration. Livers were dissected, flash frozen in liquid Nitrogen and pulverized into powders.

mRNA Measurements.

Levels of Eg5/KSP mRNA were measured in livers from all treatment groups. Samples of each liver powder (approximately ten milligrams) were homogenized in tissue lysis buffer containing proteinase K. Levels of Eg5/KSP and GAPDH mRNA were measured in triplicate for each sample using the Quantigene branched DNA assay (GenoSpectra). Mean values for Eg5/KSP were normalized to mean GAPDH values for each sample. Group means were determined and normalized to the PBS group for each experiment.

Statistical Analysis.

Significance was determined by ANOVA followed by the Tukey post-hoc test.

Results

Data Summary Mean values (±standard deviation) for Eg5/KSP mRNA are given. Statistical significance (p value) versus the PBS group is shown (ns, not significant [p>0.05]).

TABLE 8 Experiment 1 KSP/GAPDH p value PBS  1.0 ± 0.47 AD6248 10 mg/kg 0.47 ± 0.12 <0.001 unspec 10 mg/kg  1.0 ± 0.26 ns

A statistically significant reduction in liver Eg5/KSP mRNA was obtained following treatment with formulated AD6248 at a dose of 10 mg/kg.

Example 5 Silencing of Rat Liver VEGF Following Intravenous Infusion of LNP01 Formulated VSP

A “lipidoid” formulation comprising an equimolar mixture of two siRNAs was administered to rats. As used herein, VSP refers to a composition having two siRNAs, one directed to Eg5/KSP and one directed to VEGF. For this experiment the duplex AD3133 directed towards VEGF and AD12115 directed towards Eg5/KSP were used. Since Eg5/KSP expression is nearly undetectable in the adult rat liver, only VEGF levels were measured following siRNA treatment.

siRNA Duplexes Administered (VSP)

Duplex ID Target Sense Antisense AD12115 Eg5/ ucGAGAAucuAAA AGUuAGUUuAGAUU KSP cuAAcuTsT CUCGATsT (SEQ ID NO: 1240) (SEQ ID NO: 1241) AD3133 VEGF GcAcAuAGGAGAG AAGCUcAUCUCUCC AuGAGCUsU uAuGuGCusG (SEQ ID NO: 1242) (SEQ ID NO: 1243)

Key: A,G,C,U-ribonucleotides; c,u-2′-O-Me ribonucleotides; s-phosphorothioate.

Unmodified versions of each strand and the targets for each siRNA are as follows

Eg5/KSP unmod  5′ UCGAGAAUCUAAACUAACUTT 3′ sense SEQ ID NO: 1534 unmod  3′ TTAGUCCUUAGAUUUGAUUGA 5′ antisense SEQ ID NO: 1535 target 5′ UCGAGAAUCUAAACUAACU 3′ SEQ ID NO: 1311 VEGF unmod  5′ GCACAUAGGAGAGAUGAGCUU 3′ sense SEQ ID NO: 1536 unmod  3′ GUCGUGUAUCCUCUCUACUCGAA 5′ antisense SEQ ID NO: 1537 target 5′ GCACAUAGGAGAGAUGAGCUU 3′ SEQ ID NO: 1538

Methods

Dosing of Animals.

Adult, female Sprague-Dawley rats were administered lipidoid (“LNP01”) formulated siRNA by a two-hour infusion into the femoral vein. Groups of four animals received doses of 5, 10 and 15 milligrams per kilogram (mg/kg) bodyweight of formulated siRNA. Dose level refers to the total amount of siRNA duplex administered in the formulation. A fourth group received phosphate-buffered saline. Animals were sacrificed 72 hours after the end of the siRNA infusion. Livers were dissected, flash frozen in liquid Nitrogen and pulverized into powders.

Formulation Procedure

The lipidoid ND98.4HCl (MW 1487) (Formula 1, above), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) were used to prepare lipid-siRNA nanoparticles. Stock solutions of each in ethanol were prepared: ND98, 133 mg/mL; Cholesterol, 25 mg/mL, PEG-Ceramide C16, 100 mg/mL. ND98, Cholesterol, and PEG-Ceramide C16 stock solutions were then combined in a 42:48:10 molar ratio. Combined lipid solution was mixed rapidly with aqueous siRNA (in sodium acetate pH 5) such that the final ethanol concentration was 35-45% and the final sodium acetate concentration was 100-300 mM. Lipid-siRNA nanoparticles formed spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture was in some cases extruded through a polycarbonate membrane (100 nm cut-off) using a thermobarrel extruder (Lipex Extruder, Northern Lipids, Inc). In other cases, the extrusion step was omitted. Ethanol removal and simultaneous buffer exchange was accomplished by either dialysis or tangential flow filtration. Buffer was exchanged to phosphate buffered saline (PBS) pH 7.2.

Characterization of Formulations

Formulations prepared by either the standard or extrusion-free method are characterized in a similar manner. Formulations are first characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles are measured by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be 20-300 nm, and ideally, 40-100 nm in size. The particle size distribution should be unimodal. The total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA is incubated with the RNA-binding dye Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, 0.5% Triton-X100. The total siRNA in the formulation is determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The preferred range is about at least 50 nm to about at least 110 nm, preferably about at least 60 nm to about at least 100 nm, most preferably about at least 80 nm to about at least 90 nm. In one example, each of the particle size comprises at least about 1:1 ratio of Eg5 dsRNA to VEGF dsRNA.

mRNA Measurements.

Samples of each liver powder (approximately ten milligrams) were homogenized in tissue lysis buffer containing proteinase K. Levels of VEGF and GAPDH mRNA were measured in triplicate for each sample using the Quantigene branched DNA assay (GenoSpectra). Mean values for VEGF were normalized to mean GAPDH values for each sample. Group means were determined and normalized to the PBS group for each experiment.

Protein Measurements.

Samples of each liver powder (approximately 60 milligrams) were homogenized in 1 ml RIPA buffer. Total protein concentrations were determined using the Micro BCA protein assay kit (Pierce). Samples of total protein from each animal was used to determine VEGF protein levels using a VEGF ELISA assay (R&D systems). Group means were determined and normalized to the PBS group for each experiment.

Statistical Analysis.

Significance was determined by ANOVA followed by the Tukey post-hoc test

Results

Data Summary

Mean values (±standard deviation) for mRNA (VEGF/GAPDH) and protein (rel. VEGF) are shown for each treatment group. Statistical significance (p value) versus the PBS group for each experiment is shown.

TABLE 9 VEGF/GAPDH p value rel VEGF p value PBS  1.0 ± 0.17  1.0 ± 0.17  5 mg/kg 0.74 ± 0.12 <0.05  0.23 ± 0.03 <0.001 10 mg/kg 0.65 ± 0.12 <0.005 0.22 ± 0.03 <0.001 15 mg/kg 0.49 ± 0.17 <0.001 0.20 ± 0.04 <0.001

Statistically significant reductions in liver VEGF mRNA and protein were measured at all three siRNA dose levels.

Example 6 Assessment of VSP SNALP in Mouse Models of Human Hepatic Tumors

These studies utilized a VSP siRNA cocktail containing dsRNAs targeting KSP/Eg5 and dsRNAs targeting VEGF. As used herein, VSP refers to a composition having two siRNAs, one directed to Eg5/KSP and one directed to VEGF. For this experiment the duplexes AD3133 (directed towards VEGF) and AD12115 (directed towards Eg5/KSP) were used. The siRNA cocktail was formulated in SNALPs.

The maximum study size utilized 20-25 mice. To test the efficacy of the siRNA SNALP cocktail to treat liver cancer, 1×10̂6 tumor cells were injected directly into the left lateral lobe of test mice. The incisions were closed by sutures, and the mice allowed to recover for 2-5 hours. The mice were fully recovered within 48-72 hours. The SNALP siRNA treatment was initiated 8-11 days after tumor seeding.

The SNALP formulations utilized were (i) VSP (KSP+VEGF siRNA cocktail (1:1 molar ratio)); (ii) KSP (KSP+Luc siRNA cocktail); and (iii) VEGF (VEGF+Luc siRNA cocktail). All formulations contained equal amounts (mg) of each active siRNA. All mice received a total siRNA/lipid dose, and each cocktail was formulated into 1:57 cDMA SNALP (1.4% PEG-cDMA; 57.1% DLinDMA; 7.1% DPPC; and 34.3% cholesterol), 6:1 lipid:drug using original citrate buffer conditions.

Human Hep3B Study A: Anti-Tumor Activity of VSP-SNALP

Human Hepatoma Hep3B tumors were established in scid/beige mice by intrahepatic seeding. Group A (n=6) animals were administered PBS; Group B (n=6) animals were administered VSP SNALP; Group C (n=5) animals were administered KSP/Luc SNALP; and Group D (n=5) animals were administered VEGF/Luc SNALP.

SNALP treatment was initiated eight days after tumor seeding. The SNALP was dosed at 3 mg/kg total siRNA, twice weekly (Monday and Thursday), for a total of six doses (cumulative 18 mg/kg siRNA). The final dose was administered at day 25, and the terminal endpoint was at day 27.

Tumor burden was assayed by (a) body weight; (b) liver weight; (c) visual inspection+photography at day 27; (d) human-specific mRNA analysis; and (e) blood alpha-fetoprotein levels measured at day 27.

Table 10 below illustrates the results of visual scoring of tumor burden measured in the seeded (left lateral) liver lobe. Score: “−”=no visible tumor; “+”=evidence of tumor tissue at injection site; “++”=Discrete tumor nodule protruding from liver lobe; “+++”=large tumor protruding on both sides of liver lobe; “++++”=large tumor, multiple nodules throughout liver lobe.

TABLE 10 Mouse Tumor Burden Group A: PBS, day 27 1 ++++ 2 ++++ 3 ++ 4 +++ 5 ++++ 6 ++++ Group B: VSP 1 + (VEGF + KSP/Eg5, d. 27 2 − 3 − 4 − 5 ++ 6 − Group C: KSP 1 + (Luc + KSP), d. 27 2 ++ 3 − 4 + 5 ++ Group D: VEGF 1 ++++ (Luc + VEGF), d. 27 2 − 3 ++++ 4 +++ 5 ++++

Liver weights, as percentage of body weight, are shown in FIG. 1.

Body weights are shown in FIGS. 2A-2D.

From this study, the following conclusions were made. (1) VSP SNALP demonstrated potent anti-tumor effects in Hep3B 1H model; (2) the anti-tumor activity of the VSP cocktail appeared largely associated with the KSP component; (3) anti-KSP activity was confirmed by single dose histological analysis; and (4) VEGF siRNA showed no measurable effect on inhibition of tumor growth in this model.

Human Hep3B Study B: Prolonged Survival with VSP Treatment

In a second Hep3B study, human hepatoma Hep3B tumors were established by intrahepatic seeding into scid/beige mice. These mice were deficient for lymphocytes and natural killer (NK) cells, which is the minimal scope for immune-mediated anti-tumor effects. Group A (n=6) mice were untreated; Group B (n=6) mice were administered luciferase (luc) 1955 SNALP (Lot No. AP10-02); and Group C (n=7) mice were administered VSP SNALP (Lot No. AP10-01). SNALP was 1:57 cDMA SNALP, and 6:1 lipid:drug.

SNALP treatment was initiated eight days after tumor seeding. SNALP was dosed at 3 mg/kg siRNA, twice weekly (Mondays and Thursdays), for a total of six doses (cumulative 18 mg/kg siRNA). The final dose was delivered at day 25, and the terminal endpoint of the study was at day 27.

Tumor burden was assayed by (1) body weight; (2) visual inspection+photography at day 27; (3) human-specific mRNA analysis; and (4) blood alpha-fetoprotein measured at day 27.

Body weights were measured at each day of dosing (days 8, 11, 14, 18, 21, and 25) and on the day of sacrifice (FIG. 3).

TABLE 11 Tumor Burden by macroscopic Mouse observation Group A: untreated, A1R ++ day 27 A1G ++++ A1W − A2R ++++ A2G +++ A2W ++++ Group B: B1R ++++ 1955 Luc SNALP, day 27 B1G ++++ B1W +++ B2R ++ B2G +++ B2W ++++ Group C: C1R − VSP SNALP, day 27 C1G − C1B − C1W + C2R + C2G + C2W − Score: “−” = no visible tumor; “+” = evidence of tumor tissue at injection site; “++” = Discrete tumor nodule protruding from liver lobe; “+++” = large tumor protruding on both sides of liver lobe; “++++” = large tumor, multiple nodules throughout liver lobe.

The correlation between body weights and tumor burden are shown in FIGS. 4, 5 and 6.

A single dose of VSP SNALP (2 mg/kg) to Hep3B mice also resulted in the formation of mitotic spindles in liver tissue samples examined by histological staining.

Tumor burden was quantified by quantitative RT-PCR (pRT-PCR) (Taqman). Human GAPDH was normalized to mouse GAPDH via species-specific Taqman assays. Tumor score as shown by macroscopic observation in the table above correlated with GADPH levels (FIG. 7A).

Serum ELISA was performed to measure alpha-fetoprotein (AFP) secreted by the tumor. As described below, if levels of AFP go down after treatment, the tumor is not growing. Treatment with VSP lowered AFP levels in some animals compared to treatment with controls (FIG. 7B).

Human HepB3 Study C:

In a third study, human HCC cells (HepB3) were injected directly into the liver of SCID/beige mice, and treatment was initiated 20 days later. Group A animals were administered PBS; Group B animals were administered 4 mg/kg Luc-1955 SNALP; Group C animals were administered 4 mg/kg SNALP-VSP; Group D animals were administered 2 mg/kg SNALP-VSP; and Group E animals were administered 1 mg/kg SNALP-VSP. Treatment was with a single intravenous (iv) dose, and mice were sacrificed 24 hr. later.

Tumor burden and target silencing was assayed by qRT-PCR (Taqman). Tumor score was also measured visually as described above, and the results are shown in the following table. hGAPDH levels, as shown in FIG. 8, correlates with macroscopic tumor score as shown in the table below.

TABLE 12 Tumor Burden by macroscopic Mouse observation Group A: PBS A2 +++ A3 +++ A4 +++ Group B: 4 mg/kg B1 + Luc-1955 SNALP B2 +++ B3 +++ B4 +++ Group C: 4 mg/kg C1 ++ SNALP-VSP C2 ++ C3 ++ C4 +++ Group D: 2 mg/kg D1 ++ SNALP-VSP D2 + D3 + D4 ++ Group E: 1 mg/kg E1 +++ SNALP-VSP E2 + E3 ++ E4 + Score: “+” = variable tumor take/some small tumors; “++” = Discrete tumornodule protruding from liver lobe; “+++” = large tumor protruding on both sides of liver lobe

Human (tumor-derived) KSP silencing was assayed by Taqman analysis and the results are shown in FIG. 10. hKSP expression was normalized to hGAPDH. About 80% tumor KSP silencing was observed at 4 mg/kg SNALP-VSP, and efficacy was evident at 1 mg/kg. The clear bars in FIG. 9 represent the results from small (low GAPDH) tumors.

Human (tumor-derived) VEGF silencing was assayed by Taqman analysis and the results are shown in FIG. 10. hVEGF expression was normalized to hGAPDH. About 60% tumor VEGF silencing was observed at 4 mg/kg SNALP-VSP, and efficacy was evident at 1 mg/kg. The clear bars in FIG. 10 represent the results from small (low GAPDH) tumors.

Mouse (liver-derived) VEGF silencing was assayed by Taqman analysis and the results are shown in FIG. 11A. mVEGF expression was normalized to hGAPDH. About 50% liver VEGF silencing was observed at 4 mg/kg SNALP-VSP, and efficacy was evident at 1 mg/kg.

Human HepB3 Study D: Contribution of Each dsRNA to Tumor Growth

In a fourth study, human HCC cells (HepB3) were injected directly into the liver of SCID/beige mice, and treatment was initiated 8 days later. Treatment was with intravenous (iv) bolus injections, twice weekly, for a total of six does. The final dose was administered at day 25, and the terminal endpoint was at day 27.

Tumor burden was assayed by gross histology, human-specific mRNA analysis (hGAPDH qPCR), and blood alpha-fetoprotein levels (serum AFP via ELISA).

In Study 1, Group A was treated with PBS, Group B was treated with SNALP-KSP+Luc (3 mg/kg), Group C was treated with SNALP-VEGF+Luc (3 mg/kg), and Group D was treated with ALN-VSP02 (3 mg/kg).

In Study 2, Group A was treated with PBS; Group B was treated with SNALP-KSP+Luc (1 mg/kg), Group C was treated with ALN-VSP02 (1 mg/kg).

Both GAPDH mRNA levels and serum AFP levels were shown to decrease after treatment with ALN-VSP02 (FIG. 11B).

Histology Studies:

Human hepatoma Hep3B tumors were established by intrahepatic seeding in mice. SNALP treatment was initiated 20 days after tumor seeding. Tumor-bearing mice (three per group) were treated with a single intravenous (IV) dose of (i) VSP SNALP or (ii) control (Luc) SNALP at 2 mg/kg total siRNA.

Liver/tumor samples were collected for conventional H&E histology 24 hours after single SNALP administration.

Large macroscopic tumor nodules (5-10 mm) were evident at necroscopy.

Effect of ALN-VSP in Hep3B Mice:

ALN-VSP (a cocktail of KSP dsRNA and VEGF dsRNA) treatment reduced tumor burden and expression of tumor-derived KSP and VEGF. GAPDH mRNA levels, a measure of tumor burden, were also observed to decline following administration of ALN-VSP dsRNA (see FIGS. 12A-12C). A decrease in tumor burden by visual macroscopic observation was also evident following administration of ALN-VSP.

A single IV bolus injection of ALN-VSP also resulted in mitotic spindle formation that was clearly detected in liver tissue samples from Hep3B mice. This observation indicated cell cycle arrest.

Example 7 Survival of SNALP-VSP Animals Versus SNALP-Luc Treated Animals

To test the effect of siRNA SNALP on survival rates of cancer subjects, tumors were established by intrahepatic seeding in mice and the mice were treated with SNALP-siRNA. These studies utilized a VSP siRNA cocktail containing dsRNAs targeting KSP/Eg5 and VEGF. Control was dsRNA targeting Luc. The siRNA cocktail was formulated in SNALPs.

Tumor cells (Human Hepatoma Hep3B, 1×10̂6) were injected directly into the left lateral lobe of scid/beige mice. These mice were deficient for lymphocytes and natural killer (NK) cells, which is the minimal scope for immune-mediated anti-tumor effects. The incisions were closed by sutures, and the mice allowed to recover for 2-5 hours. The mice were fully recovered within 48-72 hours.

All mice received a total siRNA/lipid intravenous (iv) dose, and each cocktail was formulated into 1:57 cDMA SNALP (1.4% PEG-cDMA; 57.1% DLinDMA; 7.1% DPPC; and 34.3% cholesterol), 6:1 lipid:drug using original citrate buffer conditions.

siRNA-SNALP treatment was initiated on the day indicated below (18 or 26 days) after tumor seeding. siRNA-SNALP were administered twice a week for three weeks after 18 or 26 days at a dose of 4 mg/kg. Survival was monitored and animals were euthanized based on humane surrogate endpoints (e.g., animal body weight, abdominal distension/discoloration, and overall health).

The survival data for treatment initiated 18 days after tumor seeing is summarized in Table 13, Table 14, and FIG. 13A.

TABLE 13 Kaplan-Meier (survival) data (% Surviving) SNALP- SNALP- Day Luc VSP 18 100%  100% 22 100%  100% 25 100%  100% 27 100%  100% 28 100%  100% 28 86% 100% 29 86% 100% 32 86% 100% 33 86% 100% 33 43% 100% 35 43% 100% 36 43% 100% 36 29% 100% 38 29% 100% 38 14% 100% 38 14%  88% 40 14%  88% 43 14%  88% 45 14%  88% 49 14%  88% 51 14%  88% 51 14%  50% 53 14%  50% 53 14%  25% 55 14%  25% 57 14%  25% 57  0%  0%

TABLE 14 Survival in days, for each animal. Treatment Animal group Survival 1 SNALP-Luc 28 days 2 SNALP-Luc 33 days 3 SNALP-Luc 33 days 4 SNALP-Luc 33 days 5 SNALP-Luc 36 days 6 SNALP-Luc 38 days 7 SNALP-Luc 57 days 8 SNALP-VSP 38 days 9 SNALP-VSP 51 days 10 SNALP-VSP 51 days 11 SNALP-VSP 51 days 12 SNALP-VSP 53 days 13 SNALP-VSP 53 days 14 SNALP-VSP 57 days 15 SNALP-VSP 57 days

FIG. 13A shows the mean survival of SNALP-VSP animals and SNALP-Luc treated animals versus days after tumor seeding. The mean survival of SNALP-VSP animals was extended by approximately 15 days versus SNALP-Luc treated animals.

TABLE 15 Serum alpha fetoprotein (AFP) concentration, for each animal, at a time pre-treatment and at end of treatment (concentration in ng/ml) End of pre-Rx Rx 1 SNALP-Luc 30.858 454.454 2 SNALP-Luc 10.088 202.082 3 SNALP-Luc 23.736 648.952 4 SNALP-Luc 1.696 13.308 5 SNALP-Luc 4.778 338.688 6 SNALP-Luc 15.004 826.972 7 SNALP-Luc 11.036 245.01 8 SNALP-VSP 37.514 182.35 9 SNALP-VSP 91.516 248.06 10 SNALP-VSP 25.448 243.13 11 SNALP-VSP 24.862 45.514 12 SNALP-VSP 57.774 149.352 13 SNALP-VSP 12.446 78.724 14 SNALP-VSP 2.912 9.61 15 SNALP-VSP 4.516 11.524

Tumor burden was monitored using serum AFP levels during the course of the experiment. Alpha-fetoprotein (AFP) is a major plasma protein produced by the yolk sac and the liver during fetal life. The protein is thought to be the fetal counterpart of serum albumin, and human AFP and albumin gene are present in tandem in the same transcriptional orientation on chromosome 4. AFP is found in monomeric as well as dimeric and trimeric forms, and binds copper, nickel, fatty acids and bilirubin. AFP levels decrease gradually after birth, reaching adult levels by 8-12 months. Normal adult AFP levels are low, but detectable. AFP has no known function in normal adults and AFP expression in adults is often associated with a subset of tumors such as hepatoma and teratoma. AFP is a tumor marker used to monitor testicular cancer, ovarian cancer, and malignant teratoma. Principle tumors that secrete AFP include endodermal sinus tumor (yolk sac carcinoma), neuroblastoma, hepatoblastoma, and heptocellular carcinoma. In patients with AFP-secreting tumors, serum levels of AFP often correlate with tumor size. Serum levels are useful in assessing response to treatment. Typically, if levels of AFP go down after treatment, the tumor is not growing. A temporary increase in AFP immediately following chemotherapy may indicate not that the tumor is growing but rather that it is shrinking (and releasing AFP as the tumor cells die). Resection is usually associated with a fall in serum levels. As shown in FIG. 14, tumor burden in SNALP-VSP treated animals was significantly reduced.

The experiment was repeated with SNALP-siRNA treatment at 26, 29, 32 35, 39, and 42 days after implantation. The data is shown in FIG. 13B. The mean survival of SNALP-VSP animals was extended by approximately 15 days versus SNALP-Luc treated animals by approximately 19 days, or 38%.

Example 8 Induction of Mono-Asters in Established Tumors

Inhibition of KSP in dividing cells leads to the formation of mono asters that are readily observable in histological sections. To determine whether mono aster formation occurred in SNALP-VSP treated tumors, tumor bearing animals (three weeks after Hep3B cell implantation) were administered 2 mg/kg SNALP-VSP via tail vein injection. Control animals received 2 mg/kg SNALP-Luc. Twenty four hours later, animals were sacrificed, and tumor bearing liver lobes were processed for histological analysis. Representative images of H&E stained tissue sections are shown in FIG. 15. Extensive mono aster formation was evident in ALN VSP02 treated (A), but not SNALP-Luc treated (B), tumors. In the latter, normal mitotic figures were evident. The generation of mono asters is a characteristic feature of KSP inhibition and provides further evidence that SNALP-VSP has significant activity in established liver tumors.

Example 9 Manufacturing Process and Product Specification of ALN-VSP02 (SNALP-VSP)

ALN-VSP02 product contains 2 mg/mL of drug substance ALN-VSPDS01 formulated in a sterile lipid particle formulation (referred to as SNALP) for IV administration via infusion. Drug substance ALN-VSPDS01 consists of two siRNAs (ALN-12115 targeting KSP and ALN-3133 targeting VEGF) in an equimolar ratio. The drug product is packaged in 10 mL glass vials with a fill volume of 5 mL.

The following terminology is used herein:

Drug Substance siRNA Duplexes Single Strand Intermediates ALN-VSPDS01 ALN-12115* Sense: A-19562 Antisense: A-19563 ALN-3133** Sense: A-3981 Antisense: A-3982 *Alternate names = AD-12115, AD12115; ** Alternate names = AD-3133, AD3133

9.1 Preparation of Drug Substance ALN-VSPDS01

The two siRNA components of drug substance ALN-VSPDS01, ALN-12115 and ALN-3133, are chemically synthesized using commercially available synthesizers and raw materials. The manufacturing process consists of synthesizing the two single strand oligonucleotides of each duplex (A 19562 sense and A 19563 antisense of ALN 12115 and A 3981 sense and A 3982 antisense of ALN 3133) by conventional solid phase oligonucleotide synthesis using phosphoramidite chemistry and 5′ 0 dimethoxytriphenylmethyl (DMT) protecting group with the 2′ hydroxyl protected with tert butyldimethylsilyl (TBDMS) or the 2′ hydroxyl replaced with a 2′ methoxy group (2′ OMe). Assembly of an oligonucleotide chain by the phosphoramidite method on a solid support such as controlled pore glass or polystyrene. The cycle consists of 5′ deprotection, coupling, oxidation, and capping. Each coupling reaction is carried out by activation of the appropriately protected ribo, 2′ OMe, or deoxyribonucleoside amidite using 5 (ethylthio) 1H tetrazole reagent followed by the coupling of the free 5′ hydroxyl group of a support immobilized protected nucleoside or oligonucleotide. After the appropriate number of cycles, the final 5′ protecting group is removed by acid treatment. The crude oligonucleotide is cleaved from the solid support by aqueous methylamine treatment with concomitant removal of the cyanoethyl protecting group as well as nucleobase protecting groups. The 2′ O TBDMS group is then cleaved using a hydrogen fluoride containing reagent to yield the crude oligoribonucleotide, which is purified using strong anion exchange high performance liquid chromatography (HPLC) followed by desalting using ultrafiltration. The purified single strands are analyzed to confirm the correct molecular weight, the molecular sequence, impurity profile and oligonucleotide content, prior to annealing into the duplexes. The annealed duplex intermediates ALN 12115 and ALN 3133 are either lyophilized and stored at 20° C. or mixed in 1:1 molar ratio and the solution is lyophilized to yield drug substance ALN VSPDS01. If the duplex intermediates were stored as dry powder, they are redissolved in water before mixing. The equimolar ratio is achieved by monitoring the mixing process by an HPLC method.

The manufacturing process flow diagram is shown in FIG. 16.

Example specifications are shown in Table 16a.

The results of up to 12 month stability testing for ALN-VSPDS01 drug substance are shown in Tables 16c. The assay methods were chosen to assess physical property (appearance, pH, moisture), purity (by SEC and denaturing anion exchange chromatography) and potency (by denaturing anion exchange chromatography [AX-HPLC]).

TABLE 16a Example specifications for ALN-VSPDS01 Test Method Acceptance Criteria Appearance Visual White to off-white powder Identity, ALN-VSPDS01 Duplex AX-HPLC Duplex retention times are consistent ALN-3133 with those of reference standards ALN-12115 Identity, ALN-VSPDS01 MS Molecular weight of single strands are within the following ranges:  A-3981: 6869-6873 Da  A-3982: 7305-7309 Da A-19562: 6762-6766 Da A-19563: 6675-6679 Da Sodium counter ion (% w/w Flame AAS or ICP-OES Report data on anhydrous basis) ALN-VSPDS01 assay Denaturing AX-HPLC 90-110% Purity of ALN-VSPDS01 SEC ≧90.0 area % Single strand purity, Denaturing AX-HPLC Report data ALN-VSPDS01 Report area % for total impurities siRNA molar ratio Duplex AX-HPLC 1.0 ± 0.1 Moisture content Karl Fischer titration  ≦15% Residual solvents HS-Capillary GC ≦410 ppm Acetonitrile ≦5000 ppm Ethanol ≦5000 ppm Isopropanol pH of 1% solution USP <791> Report data Heavy metals ICP-MS Report data As, Cd, Cu, Cr, Fe, Ni, Pb, Sn Bacterial endotoxins USP <85>  ≦0.5 EU/mg Bioburden Modified USP <61> <100 CFU/g

TABLE 16b Stability of drug substance Study Storage Conditions: −20° C. (Storage Condition) Lot No.: A05M07001N Results Test Method Acceptance Criteria Initial 1 Month 3 Months 6 Months 12 Months Appearance Visual White to off- Pass Pass Pass Pass Pass white powder pH USP <791> Report data 6.7 6.4 6.6 6.4 6.8 Moisture Karl Fischer ≦15% 3.6* 6.7 6.2 5.6 5.0 content titration (% w/w) Purity SEC ≧90.0 area % 95 95 94 92 95 (area %) A-3981 Denaturing AX- Report data 24 23 23 23 23 (sense) HPLC (area %) A-3982 Denaturing AX- Report data 23 23 23 23 24 (antisense) HPLC (area %) A-19562 Denaturing AX- Report data 22 21 21 21 21 (sense) HPLC (area %) A-19563 Denaturing AX- Report data 23 22 22 22 22 (antisense) HPLC (area %)

9.2 Preparation of Drug Product ALN-VSP02 (SNALP-VSP)

ALN VSP02, is a sterile formulation of the two siRNAs (in a 1:1 molar ratio) with lipid excipients in isotonic buffer. The lipid excipients associate with the two siRNAs, protect them from degradation in the circulatory system, and aid in their delivery to the target tissue. The specific lipid excipients and the quantitative proportion of each (shown in Table 17) have been selected through an iterative series of experiments comparing the physicochemical properties, stability, pharmacodynamics, pharmacokinetics, toxicity and product manufacturability of numerous different formulations. The excipient DLinDMA is a titratable aminolipid that is positively charged at low pH, such as that found in the endosome of mammalian cells, but relatively uncharged at the more neutral pH of whole blood. This feature facilitates the efficient encapsulation of the negatively charged siRNAs at low pH, preventing formation of empty particles, yet allows for adjustment (reduction) of the particle charge by replacing the formulation buffer with a more neutral storage buffer prior to use. Cholesterol and the neutral lipid DPPC are incorporated in order to provide physicochemical stability to the particles. The polyethyleneglycol lipid conjugate PEG2000 C DMA aids drug product stability, and provides optimum circulation time for the proposed use. ALN VSP02 lipid particles have a mean diameter of approximately 80-90 nm with low polydispersity values. A representative cryo transmission electron microscope (cryo TEM) image is shown in FIG. 17. At neutral pH, the particles are essentially uncharged, with Zeta Potential values of less than 6 mV. There is no evidence of empty (non loaded) particles based on the manufacturing process.

TABLE 17 Quantitative Composition of ALN-VSP02 Component, grade Proportion (mg/mL) ALN-VSPDS01, cGMP  2.0* DLinDMA 7.3 (1,2-Dilinoleyloxy- N,N-dimethyl-3-aminopropane), cGMP DPPC (R-1,2-Dipalmitoyl-sn-glycero-3- 1.1 phosphocholine), cGMP Cholesterol, Synthetic, cGMP 2.8 PEG2000-C-DMA 0.8 (3-N-[(ω-Methoxy poly(ethylene glycol) 2000) carbamoyl]-1,2-dimyristyloxy-propylamine), cGMP Phosphate Buffered Saline, cGMP q.s. *The 1:1 molar ratio of the two siRNAs in the drug product is maintained throughout the size distribution of the drug product particles.

Solutions of lipid (in ethanol) and ALN VSPDS01 drug substance (in aqueous buffer) are mixed and diluted to form a colloidal dispersion of siRNA lipid particles with an average particle size of approximately 80-90 nm. This dispersion is then filtered through 0.45/0.2 μm filters, concentrated, and diafiltered by Tangential Flow Filtration. After in process testing and concentration adjustment to 2.0 mg/mL, the product is sterile filtered, aseptically filled into glass vials, stoppered, capped and placed at 5±3° C. The ethanol and all aqueous buffer components are USP grade; all water used is USP Sterile Water For Injection grade. Representative ALN-VSP02 process is shown in flow diagram in FIG. 18.

TABLE 18a Example ALN-VSP02 specifications Test Analytical Procedure Acceptance Criteria Appearance Visual White to off-white, homogeneous opalescent liquid, no foreign particles pH USP <791> 6.8-7.8 Osmolality USP <785> 250-350 mOsm/kg Identity, ALN-VSPDS01 Duplex Retention times consistent with ALN-3133 Anion Exchange (AX)-HPLC those of reference standards ALN-12115 Identity, ALN-VSPDS01 Denaturing Retention times consistent with A-3981 AX-HPLC those of reference standards A-3982 A-19562 A-19563 Lipid identity Reversed Phase (RP)-HPLC with Retention times consistent with DLinDMA Evaporative Light Scattering those of reference standards PEG₂₀₀₀-C-DMA (ELS) detection DPPC Cholesterol ALN-VSPDS01 label claim Denaturing 1.7-2.3 mg/mL (Strength/Potency) AX-HPLC Duplex molar ratio Duplex 1.0 ± 0.1 AX-HPLC DLinDMA content RP-HPLC with 5.6-10.3 mg/mL ELS detection PEG₂₀₀₀-C-DMA content RP-HPLC with 0.6-1.1 mg/mL ELS detection DPPC content RP-HPLC with 0.8-1.5 mg/mL ELS detection Cholesterol content RP-HPLC with 2.1-3.9 mg/mL ELS detection Total lipid: ALN-VSPDS01 ratio Calculated from total lipid 4.9-8.1 mg/mg assay and label claim for drug substance ALN-VSPDS01 encapsulation Fluorometric assay ≧90.0% Purity Denaturing ≧80.0 area % AX-HPLC Impurity profile Denaturing Report retention times (relative to AX-HPLC A-19563) and area % for all peaks ≧0.20% Residual ethanol USP <467> ≦5000 ppm Residual EDTA Ion Pairing (IP) HPLC with ≦2000 μg/mL UV detection Particle size Z average Dynamic light scattering 60-120 nm Polydispersity Dynamic light scattering ≦0.15 Particle size distribution Dynamic light scattering Report data D₁₀ D₅₀ D₉₀ Particulate matter Modified USP <788> ≦300 per container ≧25 μm ≦3000 per container ≧10 μm Bacterial endotoxins Modified USP <85>  ≦5.0 EU/mL Sterility USP <71>  Pass Volume in container USP <1>  ≧5.0 mL Dose uniformity USP <905> Pass Heavy metal analysis Inductive Coupled Plasma Mass Report data Spectrometry (ICP-MS)

9.4 Container/Closure System

The ALN VSP02 drug product is packaged in 10 mL glass vials with a fill volume of 5 mL. The container closure system is comprised of a USP/EP Type I borosilicate glass vial, a teflon faced butyl rubber stopper and an aluminum flip off cap. The drug product will be stored at 5±3° C.

9.5 Stability of Drug Product ALN-VSP02

Stability data (25° C./60% RH) are given in Table 18b and 18c.

TABLE 18b Example ALN-VSP02 stability at storage conditions Study Storage Conditions: 2-8° C. Lot No.: IC097 Results Test Method Acceptance Criteria Initial 1 Month 2 Months 3 Months 4 Months 6 Months Appearance Visual White to off-white, Pass Pass Pass Pass Pass Pass homogeneous opalescent liquid, no foreign particles pH USP <791> 6.8-7.8 7.4 7.4 7.4 7.3 7.4 7.3 Osmolality USP <785> 250-350 mOsm/kg 308 307 305 306 309 305 ALN- Duplex Retention times Pass Pass Pass Pass Pass Pass VSPDS01 AX-HPLC consistent with Identity, those of reference ALN-3133 standards ALN- 12115 ALN- Denaturing Retention times Pass Pass Pass Pass Pass Pass VSPDS01 AX-HPLC consistent with Identity, those of reference A-3981 standards A-3982 A-19562 A-19563 Lipid RP-HPLC Retention times Pass Pass Pass Pass Pass Pass identity, with ELS consistent with DLinDMA Detection those of reference PEG₂₀₀₀- standards C-DMA DPPC Cholesterol ALN- Denaturing 1.7-2.3 mg/mL 2.1 2.2 2.1 2.1 2.1 2.1 VSPDS01 AX-HPLC strength/potency Duplex Duplex 1.0 ± 0.1 1.0 1.0 1.0 1.0 1.0 1.0 molar ratio AX-HPLC DLinDMA RP-HPLC 5.6-10.3 mg/mL 9.1 9.4 9.1 9.6 9.1 9.2 content with ELS Detection Cholesterol RP-HPLC 2.1-3.9 mg/mL 3.4 3.5 3.4 3.5 3.4 3.5 content with ELS Detection DPPC content RP-HPLC 0.8-1.5 mg/mL 1.3 1.3 1.4 1.4 1.2 1.3 with ELS Detection PEG₂₀₀₀- RP-HPLC 0.6-1.1 mg/mL 1.0 1.0 1.0 1.1 1.0 1.0 C-DMA with ELS content Detection Total Calculation 4.9-8.1 mg/mg 7.0 6.9 7.1 7.4 7.0 7.1 lipid:ALN- VSPDS01 ratio ALN- Fluorometric ≧90.0% 95.9 96.5 94.4 98.1 97.8 96.4 VSPDS01 assay encapsulation Purity Denaturing ≧80.0% 90.7 89.6 90.8 91.3 92.4 90.8 AX-HPLC Particle size, Light 60-120 nm 86 87 87 87 87 87 Z-average scattering Polydispersity Light ≦0.15 0.02 0.03 0.02 0.03 0.03 0.03 scattering Particle size Light Report 56 56 56 56 56 56 distribution, scattering data (nm) D₁₀ Particle size Light Report 76 77 77 77 78 77 distribution, scattering data (nm) D₅₀ Particle size Light Report 110 112 112 113 112 113 distribution, scattering data (nm) D₉₀ Particulate matter, Modified (per container) NS NS NS NS ≧25 μm USP <788> ≦300 18 3 ≧10 μm ≦3000 48 11 Bacterial USP <85> ≦5.0 EU/mL 0.50 NS NS NS NS NS endotoxins Sterility USP <71> Pass Pass NS NS NS NS NS

TABLE 18c Example ALN-VSP02 stability at 25° C./ambient humidity Study Storage Conditions: 25° C./ambient humidity Lot No.: IC097 Results Test Method Acceptance Criteria Initial 1 Month 2 Months 3 Months 4 Months 6 Months Appearance Visual White to off-white, Pass Pass Pass Pass Pass Pass homogeneous opalescent liquid, no foreign particles pH USP <791> 6.8-7.8 7.4 7.3 7.2 7.1 7.2 7.1 Osmolality USP <785> 250-350 mOsm/kg 308 306 304 307 307 304 ALN- Duplex Retention times Pass Pass Pass Pass Pass Pass VSPDS01 AX-HPLC consistent with Identity, those of reference ALN-3133 standards ALN- 12115 ALN- Denaturing Retention times Pass Pass Pass Pass Pass Pass VSPDS01 AX-HPLC consistent with Identity, those of reference A-3981 standards A-3982 A-19562 A-19563 Lipid RP-HPLC Retention times Pass Pass Pass Pass Pass Pass identity, with ELS consistent with DLinDMA Detection those of reference PEG₂₀₀₀- standards C-DMA DPPC Cholesterol ALN- Denaturing 1.7-2.3 mg/mL 2.1 2.1 2.0 2.0 2.0 2.0 VSPDS01 AX-HPLC strength/ potency Duplex Duplex 1.0 ± 0.1 1.0 1.0 1.0 1.0 1.0 1.0 molar ratio AX-HPLC DLinDMA RP-HPLC 5.6-10.3 mg/mL 9.1 9.6 9.0 9.3 9.2 9.3 content with ELS Detection Cholesterol RP-HPLC 2.1-3.9 mg/mL 3.4 3.5 3.4 3.5 3.4 3.5 content with ELS Detection DPPC RP-HPLC 0.8-1.5 mg/mL 1.3 1.3 1.3 1.2 1.2 1.1 content with ELS Detection PEG₂₀₀₀- RP-HPLC 0.6-1.1 mg/mL 1.0 1.0 1.0 1.1 1.0 1.0 C-DMA with ELS content Detection Total Calculation 4.9-8.1 mg/mg 7.0 7.3 7.4 7.6 7.4 7.5 lipid:ALN- VSPDS01 ratio ALN- Fluorometric ≧90.0% 95.9 97.2 94.6 97.9 97.9 96.7 VSPDS01 assay encapsulation Purity Denaturing ≧80.0% 90.7 88.0 88.9 88.4 89.0 85.3 AX-HPLC Particle size, Light 60-120 nm 86 85 86 89 87 87 Z-average scattering Polydispersity Light ≦0.15 0.02 0.05 0.03 0.04 0.04 0.03 scattering Particle size Light Report 56 54 56 58 56 57 distribution, scattering data (nm) D₁₀ Particle size Light Report 76 75 77 79 77 78 distribution, scattering data (nm) D₅₀ Particle size Light Report 110 110 111 116 113 113 distribution, scattering data (nm) D₉₀ Particulate matter, Modified (per container) NS NS NS NS ≧25 μm USP <788> ≦300 18 1 ≧10 μm ≦3000 48 16 Bacterial USP <85> ≦5.0 EU/mL 0.50 NS NS NS NS <0.50 endotoxins Sterility USP <71> Pass Pass NS NS NS NS Pass

Example 10 In Vitro Efficacy of ALN-VSP02 in Human Cancer Cell Lines

The efficacy of ALN-VSP02 treatment in human cancer cell lines was determined via measurement of KSP mRNA, VEGF mRNA, and cell viability after treatment. IC50 (nM) values determined for KSP and VEGF in each cell line.

TABLE 19 cell lines Cell line tested ATCC cat number HELA ATCC Cat N: CCL-2 KB ATCC Cat N: CCL-17 HEP3B ATCC Cat N: HB-8064 SKOV-3 ATCC Cat N: HTB-77 HCT-116 ATCC Cat N: CCL-247 HT-29 ATCC Cat N: HTB-38 PC-3 ATCC Cat N: CRL-1435 A549 ATCC Cat N: CCL-185 MDA-MB-231 ATCC Cat N: HTB-26

Cells were plated in 96 well plates in complete media at day 1 to reach a density of 70% on day 2. On day 2 media was replaced with Opti-MEM reduced serum media (Invitrogen Cat N: 11058-021) and cells were transfected with either ALN-VSP02 or control SNALP-Luc with concentration range starting at 1.8 μM down to 10 pM. After 6 hours the media was changed to complete media. Three replicate plates for each cell line for each experiment was done.

Cells were harvested 24 hours after transfection. KSP levels were measured using bDNA; VEGF mRNA levels were measured using human TaqMan assay.

Viability was measured using Cell Titer Blue reagent (Promega Cat N: G8080) at 48 and/or 72 h following manufacturer's recommendations.

As shown in Table 20, nM concentrations of VSP02 are effective in reducing expression of both KSP and VEGF in multiple human cell lines. Viability of treated cells was not

TABLE 20 Results IC50 (nM) IC50 (nM) Cell line KSP VEGF HeLa 8.79 672 SKOV-3 142 1347 HCT116 31.6 27.5 Hep3B 1.3 14.5 HT-29 262 ND PC3 127 ND KB 50.6 ND A549 201 ND MB231 187 ND

Example 11 Anti-Tumor Efficacy of VSP SNALP Vs. Sorafenib in Established Hep3B Intrahepatic Tumors

The anti-tumor effects of multi-dosing VSP SNALP verses Sorafenib in scid/beige mice bearing established Hep3B intrahepatic tumors was studied. Sorafenib is a small molecule inhibitor of protein kinases approved for treatment of hepatic cellular carcinoma (HCC).

Tumors were established by intrahepatic seeding in scid/beige mice as described herein. Treatment was initiated 11 days post-seeding. Mice were treated with Sorafenib and a control siRNA-SNALP, Sorafenib and VSP siRNA-SNALP, or VSP siRNA-SNALP only. Control mice were treated with buffers only (DMSO for Sorafenib and PBS for siRNA-SNALP). Sorafenib was administered intraparenterally from Mon to Fri for three weeks, at 15 mg/kg according to body weight for a total of 15 injections. Sorafenib was administered a minimum of 1 hour after SNALP injections. The siRNA-SNALPS were administered intravenously via the lateral tail vein according at 3 mg/kg based on the most recently recorded body weight (10 ml/kg) for 3 weeks (total of 6 doses) on days 1, 4, 7, 10, 14, and 17.

Mice were euthanized based on an assessment of tumor burden including progressive weight loss and clinical signs including condition, abdominal distension/discoloration and mobility.

The percent survival data are shown in FIG. 19. Co-administration of VSP siRNA-SNALP with Sorafenib increased survival proportion compared to administration of Sorafenib or VSP siRNA-SNALP alone. VSP siRNA-SNALP increased survival proportion compared to Sorafenib.

Example 12 In Vitro Efficacy of VSP Using Variants of AD-12115 and AD-3133

Two sets of duplexes targeted to Eg5/KSP and VEGF were designed and synthesized. Each set included duplexes tiling 10 nucleotides in each direction of the target sites for either AD-12115 and AD-3133.

Sequences of the target, sense strand, and antisense strand for each duplex are shown in the Table below.

Each duplex is assayed for inhibition of expression using the assays described herein. The duplexes are administered alone and/or in combination, e.g., an Eg5/KSP dsRNA in combination with a VEGF dsRNA. In some embodiments, the dsRNA are administered in a SNALP formulation as described herein.

TABLE 21 Sequences of dsRNA targeted to VEGF and Eg5/KSP (tiling) SEQ Sense Strand SEQ Duplex target target sequence ID Antisense strand ID ID gene 5′ to 3′ NO: 5′ to 3′ NO: AD- VEGFA ACCAAGGCCAGCACAUAGG 2264 AccAAGGccAGcAcAuAGGTsT 2304 20447.1 CCuAUGUGCUGGCCUUGGUTsT 2305 AD- VEGFA CCAAGGCCAGCACAUAGGA 2265 ccAAGGccAGcAcAuAGGATsT 2306 20448.1 UCCuAUGUGCUGGCCUUGGTsT 2307 AD- VEGFA CCAAGGCCAGCACAUAGGA 2266 ccAAGGccAGcAcAuAGGATsT 2308 20449.1 CUCCuAUGUGCUGGCCUUGTsT 2309 AD- VEGFA AAGGCCAGCACAUAGGAGA 2267 AAGGccAGcAcAuAGGAGATsT 2310 20450.1 UCUCCuAUGUGCUGGCCUUTsT 2311 AD- VEGFA AGGCCAGCACAUAGGAGAG 2268 AGGccAGcAcAuAGGAGAGTsT 2312 20451.1 CUCUCCuAUGUGCUGGCCUTsT 2313 AD- VEGFA GGCCAGCACAUAGGAGAGA 2269 GGccAGcAcAuAGGAGAGATsT 2314 20452.1 UCUCUCCuAUGUGCUGGCCTsT 2315 AD- VEGFA GCCAGCACAUAGGAGAGAU 2270 GccAGcAcAuAGGAGAGAuTsT 2316 20453.1 AUCUCUCCuAUGUGCUGGCTsT 2317 AD- VEGFA CCAGCACAUAGGAGAGAUG 2271 ccAGcAcAuAGGAGAGAuGTsT 2318 20454.1 cAUCUCUCCuAUGUGCUGGTsT 2319 AD- VEGFA CAGCACAUAGGAGAGAUGA 2272 cAGcAcAuAGGAGAGAuGATsT 2320 20455.1 UcAUCUCUCCuAUGUGCUGTsT 2321 AD- VEGFA AGCACAUAGGAGAGAUGAG 2273 AGcAcAuAGGAGAGAuGAGTsT 2322 20456.1 CUcAUCUCUCCuAUGUGCUTsT 2323 AD- VEGFA CACAUAGGAGAGAUGAGCU 2274 cAcAuAGGAGAGAuGAGcuTsT 2324 20457.1 AGCUcAUCUCUCCuAUGUGTsT 2325 AD- VEGFA ACAUAGGAGAGAUGAGCUU 2275 AcAuAGGAGAGAuGAGcuuTsT 2326 20458.1 AAGCUcAUCUCUCCuAUGUTsT 2327 AD- VEGFA CAUAGGAGAGAUGAGCUUC 2276 cAuAGGAGAGAuGAGcuucTsT 2328 20459.1 GAAGCUcAUCUCUCCuAUGTsT 2329 AD- VEGFA AUAGGAGAGAUGAGCUUCC 2277 AuAGGAGAGAuGAGcuuccTsT 2330 20460.1 GGAAGCUcAUCUCUCCuAUTsT 2331 AD- VEGFA UAGGAGAGAUGAGCUUCCU 2278 uAGGAGAGAuGAGcuuccuTsT 2332 20461.1 AGGAAGCUcAUCUCUCCuATsT 2333 AD- VEGFA AGGAGAGAUGAGCUUCCUA 2279 AGGAGAGAuGAGcuuccuATsT 2334 20462.1 uAGGAAGCUcAUCUCUCCUTsT 2335 AD- VEGFA GGAGAGAUGAGCUUCCUAC 2280 GGAGAGAuGAGcuuccuAcTsT 2336 20463.1 GuAGGAAGCUcAUCUCUCCTsT 2337 AD- VEGFA GAGAGAUGAGCUUCCUACA 2281 GAGAGAuGAGcuuccuAcATsT 2338 20464.1 UGuAGGAAGCUcAUCUCUCTsT 2339 AD- VEGFA AGAGAUGAGCUUCCUACAG 2282 AGAGAuGAGcuuccuAcAGTsT 2340 20465.1 CUGuAGGAAGCUcAUCUCUTsT 2341 AD- VEGFA GAGAUGAGCUUCCUACAGC 2283 GAGAuGAGcuuccuAcAGcTsT 2342 20466.1 GCUGuAGGAAGCUcAUCUCTsT 2343 AD- KSP AUGUUCCUUAUCGAGAAUC 2284 AuGuuccuuAucGAGAAucTsT 2344 20467.1 GAUUCUCGAuAAGGAAcAUTsT 2345 AD- KSP UGUUCCUUAUCGAGAAUCU 2285 uGuuccuuAucGAGAAucuTsT 2346 20468.1 AGAUUCUCGAuAAGGAAcATsT 2347 AD- KSP GUUCCUUAUCGAGAAUCUA 2286 GuuccuuAucGAGAAucuATsT 2348 20469.1 uAGAUUCUCGAuAAGGAACTsT 2349 AD- KSP UUCCUUAUCGAGAAUCUAA 2287 uuccuuAucGAGAAucuAATsT 2350 20470.1 UuAGAUUCUCGAuAAGGAATsT 2351 AD- KSP UCCUUAUCGAGAAUCUAAA 2288 uccuuAucGAGAAucuAAATsT 2352 20471.1 UUuAGAUUCUCGAuAAGGATsT 2353 AD- KSP CCUUAUCGAGAAUCUAAAC 2289 ccuuAucGAGAAucuAAAcTsT 2354 20472.1 GUUuAGAUUCUCGAuAAGGTsT 2355 AD- KSP CUUAUCGAGAAUCUAAACU 2290 cuuAucGAGAAucuAAAcuTsT 2356 20473.1 AGUUuAGAUUCUCGAuAAGTsT 2357 AD- KSP UUAUCGAGAAUCUAAACUA 2291 uuAucGAGAAucuAAAcuATsT 2358 20474.1 uAGUUuAGAUUCUCGAuAATsT 2359 AD- KSP UAUCGAGAAUCUAAACUAA 2292 uAucGAGAAucuAAAcuAATsT 2360 20475.1 UuAGUUuAGAUUCUCGAuATsT 2361 AD- KSP AUCGAGAAUCUAAACUAAC 2293 AucGAGAAucuAAAcuAAcTsT 2362 20476.1 GUuAGUUuAGAUUCUCGAUTsT 2363 AD- KSP CGAGAAUCUAAACUAACUA 2294 cGAGAAucuAAAcuAAcuATsT 2364 20477.1 uAGUuAGUUuAGAUUCUCGTsT 2365 AD- KSP GAGAAUCUAAACUAACUAG 2295 GAGAAucuAAAcuAAcuAGTsT 2366 20478.1 CuAGUuAGUUuAGAUUCUCTsT 2367 AD- KSP AGAAUCUAAACUAACUAGA 2296 AGAAucuAAAcuAAcuAGATsT 2368 20479.1 UCuAGUuAGUUuAGAUUCUTsT 2369 AD- KSP GAAUCUAAACUAACUAGAA 2297 GAAucuAAAcuAAcuAGAATsT 2370 20480.1 UUCuAGUuAGUUuAGAUUCTsT 2371 AD- KSP AAUCUAAACUAACUAGAAU 2298 AAucuAAAcuAAcuAGAAuTsT 2372 20481.1 AUUCuAGUuAGUUuAGAUUTsT 2373 AD- KSP AUCUAAACUAACUAGAAUC 2299 AucuAAAcuAAcuAGAAucTsT 2374 20482.1 GAUUCuAGUuAGUUuAGAUTsT 2375 AD- KSP UCUAAACUAACUAGAAUCC 2300 ucuAAAcuAAcuAGAAuccTsT 2376 20483.1 GGAUUCuAGUuAGUUuAGATsT 2377 AD- KSP CUAAACUAACUAGAAUCCU 2301 cuAAAcuAAcuAGAAuccuTsT 2378 20484.1 AGGAUUCuAGUuAGUUuAGTsT 2379 AD- KSP UAAACUAACUAGAAUCCUC 2302 uAAAcuAAcuAGAAuccucTsT 2380 20485.1 GAGGAUUCuAGUuAGUUuATsT 2381 AD- KSP AAACUAACUAGAAUCCUCC 2303 AAAcuAAcuAGAAuccuccTsT 2382 20486.1 GGAGGAUUCuAGUuAGUUUTsT 2383

Example 13 VEGF Targeted dsRNA with a Single Blunt End

A set duplexes targeted to VEGF were designed and synthesized. The set included duplexes tiling 10 nucleotides in each direction of the target sites for AD-3133. Each duplex includes a 2 base overhang at the end corresponding to the 3′ end of the antisense strand and no overhang, e.g., a blunt end, at the end corresponding to the 5′ end of the antisense strand.

The sequences of each strand of these duplexes are shown in the following table.

Each duplex is assayed for inhibition of expression using the assays described herein. The VEGF duplexes are administered alone and/or in combination with an Eg5/KSP dsRNA (e.g., AD-12115). In some embodiments, the dsRNA are administered in a SNALP formulation as described herein.

TABLE 22 Target sequences of blunt ended dsRNA targeted to VEGF SEQ position ID VEGF target on VEGF duplex ID NO: sequence 5′ to 3′ gene AD-20447.1 2384 ACCAAGGCCAGCACAUAGG 1365 AD-20448.1 2385 CCAAGGCCAGCACAUAGGA 1366 AD-20449.1 2386 CAAGGCCAGCACAUAGGAG 1367 AD-20450.1 2387 AAGGCCAGCACAUAGGAGA 1368 AD-20451.1 2388 AGGCCAGCACAUAGGAGAG 1369 AD-20452.1 2389 GGCCAGCACAUAGGAGAGA 1370 AD-20453.1 2390 GCCAGCACAUAGGAGAGAU 1371 AD-20454.1 2391 CCAGCACAUAGGAGAGAUG 1372 AD-20455.1 2392 CAGCACAUAGGAGAGAUGA 1373 AD-20456.1 2393 AGCACAUAGGAGAGAUGAG 1374 AD-20457.1 2394 CACAUAGGAGAGAUGAGCU 1376 AD-20458.1 2395 ACAUAGGAGAGAUGAGCUU 1377 AD-20459.1 2396 CAUAGGAGAGAUGAGCUUC 1378 AD-20460.1 2397 AUAGGAGAGAUGAGCUUCC 1379 AD-20461.1 2398 UAGGAGAGAUGAGCUUCCU 1380 AD-20462.1 2399 AGGAGAGAUGAGCUUCCUA 1381 AD-20463.1 2400 GGAGAGAUGAGCUUCCUAC 1382 AD-20464.1 2401 GAGAGAUGAGCUUCCUACA 1383 AD-20465.1 2402 AGAGAUGAGCUUCCUACAG 1384 AD-20466.1 2403 GAGAUGAGCUUCCUACAGC 1385

TABLE 23 Strand sequences of blunt ended dsRNA targeted to VEGF SEQ SEQ Sense strand ID Antisense strand ID duplex ID (5′ to 3′) NO: (5′ to 3′) NO: AD-20447.1 ACCAAGGCCAGCACAUAGGAG 2404 CUCCUAUGUGCUGGCCUUGGUGA 2424 AD-20448.1 CCAAGGCCAGCACAUAGGAGA 2405 UCUCCUAUGUGCUGGCCUUGGUG 2425 AD-20449.1 CAAGGCCAGCACAUAGGAGAG 2406 CUCUCCUAUGUGCUGGCCUUGGU 2426 AD-20450.1 AAGGCCAGCACAUAGGAGAGA 2407 UCUCUCCUAUGUGCUGGCCUUGG 2427 AD-20451.1 AGGCCAGCACAUAGGAGAGAU 2408 AUCUCUCCUAUGUGCUGGCCUUG 2428 AD-20452.1 GGCCAGCACAUAGGAGAGAUG 2409 CAUCUCUCCUAUGUGCUGGCCUU 2429 AD-20453.1 GCCAGCACAUAGGAGAGAUGA 2410 UCAUCUCUCCUAUGUGCUGGCCU 2430 AD-20454.1 CCAGCACAUAGGAGAGAUGAG 2411 CUCAUCUCUCCUAUGUGCUGGCC 2431 AD-20455.1 CAGCACAUAGGAGAGAUGAGC 2412 GCUCAUCUCUCCUAUGUGCUGGC 2432 AD-20456.1 AGCACAUAGGAGAGAUGAGCU 2413 AGCUCAUCUCUCCUAUGUGCUGG 2433 AD-20457.1 CACAUAGGAGAGAUGAGCUUC 2414 GAAGCUCAUCUCUCCUAUGUGCU 2434 AD-20458.1 ACAUAGGAGAGAUGAGCUUCC 2415 GGAAGCUCAUCUCUCCUAUGUGC 2435 AD-20459.1 CAUAGGAGAGAUGAGCUUCCU 2416 AGGAAGCUCAUCUCUCCUAUGUG 2436 AD-20460.1 AUAGGAGAGAUGAGCUUCCUA 2417 UAGGAAGCUCAUCUCUCCUAUGU 2437 AD-20461.1 UAGGAGAGAUGAGCUUCCUAC 2418 GUAGGAAGCUCAUCUCUCCUAUG 2438 AD-20462.1 AGGAGAGAUGAGCUUCCUACA 2419 UGUAGGAAGCUCAUCUCUCCUAU 2439 AD-20463.1 GGAGAGAUGAGCUUCCUACAG 2420 CUGUAGGAAGCUCAUCUCUCCUA 2440 AD-20464.1 GAGAGAUGAGCUUCCUACAGC 2421 GCUGUAGGAAGCUCAUCUCUCCU 2441 AD-20465.1 AGAGAUGAGCUUCCUACAGCA 2422 UGCUGUAGGAAGCUCAUCUCUCC 2442 AD-20466.1 GAGAUGAGCUUCCUACAGCAC 2423 GUGCUGUAGGAAGCUCAUCUCUC 2443

Example 14 Inhibition of Eg5/KSP and VEGF Expression in Humans

A human subject is treated with a pharmaceutical composition, e.g., ALNVSP02, having both a SNALP formulated dsRNA targeted to a Eg5/KSP gene and a SNALP formulated dsRNA targeted to a VEGF gene to inhibit expression of the Eg5/KSP and VEGF genes.

A subject in need of treatment is selected or identified. The subject can be in need of cancer treatment, e.g., liver cancer.

At time zero, a suitable first dose of the composition is subcutaneously administered to the subject. The composition is formulated as described herein. After a period of time, the subject's condition is evaluated, e.g., by measurement of tumor growth, measuring serum AFP levels, and the like. This measurement can be accompanied by a measurement of Eg5/KSP and/or VEGF expression in said subject, and/or the products of the successful siRNA-targeting of Eg5/KSP and/or VEGF mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.

After treatment, the subject's condition is compared to the condition existing prior to the treatment, or relative to the condition of a similarly afflicted but untreated subject.

Those skilled in the art are familiar with methods and compositions in addition to those specifically set out in the present disclosure which will allow them to practice this invention to the full scope of the claims hereinafter appended. 

1. A composition comprising a first double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human kinesin family member 11 (Eg5/KSP) gene in a cell and a second dsRNA for inhibiting expression of a human VEGF in a cell, wherein: both said first and said second dsRNA are formulated in a stable nucleic acid lipid particle (SNALP) comprising DLinDMA, cholesterol, DPPC, and PEG2000-C-DMA; said first dsRNA consists of a first sense strand and a first antisense strand, and said first sense strand comprises a first sequence and said first antisense strand comprises a second sequence complementary to at least 15 contiguous nucleotides of SEQ ID NO:1311 (5 ‘-UCGAGAAUCUAAACUAACU-3’), wherein said first sequence is complementary to said second sequence and wherein said first dsRNA is between 15 and 30 base pairs in length; and said second dsRNA consists of a second sense strand and a second antisense strand, said second sense strand comprising a third sequence and said second antisense strand comprising a fourth sequence complementary to at least 15 contiguous nucleotides of SEQ ID NO:1538 (5′-GCACAUAGGAGAGAUGAGCUU-3′), wherein said third sequence is complementary to said fourth sequence and wherein each strand is between 15 and 30 base pairs in length.
 2. A composition comprising a first double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human kinesin family member 11 (Eg5/KSP) gene in a cell and a second dsRNA for inhibiting expression of a human VEGF in a cell, wherein: both said first and said second dsRNA are formulated in a stable nucleic acid lipid particle (SNALP) comprising DLinDMA, cholesterol, DPPC, and PEG2000-C-DMA; said first dsRNA consists of a first sense strand and a first antisense strand, and said first sense strand comprises a first sequence and said first antisense strand comprises a second sequence complementary to at least 15 contiguous nucleotides of SEQ ID NO:1273, wherein said first sequence is complementary to said second sequence and wherein said first dsRNA is between 15 and 30 base pairs in length; and said second dsRNA consists of a second sense strand and a second antisense strand, said second sense strand comprising a third sequence and said second antisense strand comprising a fourth sequence complementary to at least 15 contiguous nucleotides of SEQ ID NO:2264, wherein said third sequence is complementary to said fourth sequence and wherein each strand is between 15 and 30 base pairs in length. 